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SOFTmax PRO User`s Manual

1. Figure 10 67 The Group Section for Data The columns in this group are set up as follows from left to right e Sample the nine sample names assigned for the Data group Wells the wells that are assigned to the individual sample names these correspond to the individual columns in the template Concentration the concentration assigned to each sample in the template e Values raw RFU values obtained for each sample e MeanValue the mean of data from the Value column for each sample e Activation sample activation relative to selected control group e CV coefficient variation percent for each sample value Viewing Formulas To view the formulas associated with all columns in the Data Group section hold down the CTRL Shift keys while this group is active A second row will appear beneath the column titles showing the for 10 57 Chapter 10 Tutorials 10 58 B mulas for each column Depending on the size of the column all or only part of the formula may be shown Lr kal HT Data Sample Wells Concentration Values MeanValue Aotivation CW Sampled fell Concentration Wela Average Value Mean valued Summ Cual Data z Az 0 0410 824 450 Bie 4920 254 58 575 9 3 Figure 10 68 A Portion of the Data Group Section with Formulas Shown by Pressing the CTRL Shift Keys If colu
2. M M Rt ttm RR RR RR D RETE E IEEE E ERR RR c RE E E a RE RETE Rm ILm1 A OS uuu bee A X A o M BO ait c a E EEE E EEE R R E RR 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 Time secs Ymax Points 14 Vell o B4 Ymax Per Second 14 267 0 970 R 2 Figure 8 10 Well Graph for a Single Kinetic Microplate Well Raw Data Multiple wells from the same Plate section can be graphed together in one enlarged window Select the well s in the data display hold the Shift key if you are selecting non contiguous wells you can drag across contiguous SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments wells and then click the Graph button or double click in one of the selected wells Pi Plate
3. 10 16 COMMOlS RIGCG ot nee ncee peda susdartot eine E E EE a 10 16 Samples with Dilution Added 0 cece cece eee eee eens 10 17 Samples with Dilution Added 0 2 eee eee eee eens 10 17 Series Dialog Box for Samples with Dilution 00 e ee ee eee 10 18 Samples with Dilution Made into a Series 0 00 cece eee eee 10 18 The Plate Section Showing Group Boundaries esee eee eee 10 19 Reduction Dialog Box Single Wavelength Endpoint 44 10 19 Display DIO DOS spors r rr EVI Soa tent irs v eases Sodr it qo LIBRE MV E 10 20 Save As Dialog Box Macintosh 0 cece cee eens 10 21 Save As Dialog Box Windows 0 0 cece eee ees 10 21 Extended Choices in the Control Menu cece eee eee eens 10 22 Plate Section with Simulated Data Absorbance instrument example 10 23 Display Did 02 DOK ausu ute u peu aure E durare du ed uud apta dnd cwn dus aat 10 23 Display Options for Reduced Displays Absorbance shown 10 24 Display Dialog Box Updated with Low and High Limit Settings Absorbance shown eee RR RR IRR es 10 24 Plate Display with Data Displayed as Gray Scale Absorbance shown 10 25 The Group Section for Standards Absorbance shown Luuse 10 25 A Portion of the Standard Group Section with Formulas Shown by Pressing the CTRL Shift Keys 000
4. 410 000 410 00d 410 0080 420 000 420 000 420 000 420 000 420 000 Smallest standard value 470 000 Largest standard value 390 000 Figure 3 13 Group Section 425 000 Group sections are created automatically when you create or select groups for the experiment in the Template Editor A Group section is a table which shows all of the information for a particular group A group is a set of associ ated samples which have been created in the Template Depending on the default assay chosen certain group types such as Standard or Unknown may have been created you can create others as required Group sections may also be created by duplicating existing Group sections The Group section is divided into three areas e The tool bar The body of the Group table e The summaries The Group menu contains the following items shown with their tool bar or mouse equivalents These menu items and buttons are active only when a Group section is active 3 19 Chapter 3 SoftMax Pro Overview l F Table 3 4 Group Section Menu Commands and Tool Bar Buttons Menu Command Description Tool Bar Button Mouse Equivalent New Window in the Opens the Group section Group menu in a new window Name
5. CuvetteSet 1 Click in the tool bar of the Endpoint active section Lmi 450 and drag upward Eef Wo Reference Wavelength Combination Lri Data Mode Absorbance Figure 9 8 Moving a Section Upward SoftMax Pro User s Manual 9 13 Chapter 9 File Management and Printing Changing the lext Format The text in Notes and Group sections can be formatted to change its appear ance The Format Text dialog box accessible by choosing Text Style 387 CTRL T from the Edit menu provides a number of different options see Figure 9 9 Format Text size IZ taie C Underline ABCDEabcde I Outline Apply 1234567690 In Shadow O Condensed i Extended Figure 9 9 Format Text Dialog Box Macintosh First highlight the text you wish to format Then choose Text Style 881 CTRL T and select the options you wish to apply to the selected text You can click on the Appl y button to see the result of the selections you have made allowing you to select more or different options before finally setting the options by clicking on OK The Print Dialog Box Choosing any of the Print commands brings up a dialog box The contents of this box will vary depending upon the type of printer you have selected or the particular printing option you have chosen but the basic elements are the same The dialog boxes shown below represent those displayed when you choose the Print com
6. Minimize a M Expand a6E Hide Status Experiment 1l b Experiment 2 gt Minimize 3 M CTRL M If you select Minimize while a section within an ex periment is active the sections within that experiment are minimized If you choose Minimize while an experiment tool bar is active all ex periments will be minimized sections within are hidden and only the experiment tool bars are visible Expand 3 E CTRL E If you select Expand while a section within an experi ment is active the sections within that experiment are expanded If you choose Expand while an experiment tool bar is active all sections within all experiments become visible the sections may be minimized or expanded Hide Status Show Status Hides or displays the status bar Experiment Menu The Experiment menu provides five commands New New Notes New Plate New CuvetteSet and New Graph New Experiment New Creates a new experiment directly following the active experiment the New Notes new experiment then becomes the active experiment All sections Mew Plate 36K shown in the experiment that was active when this command is cho New CuvetteSet sen are duplicated in the new experiment New Graph New Notes Creates a new Notes section below the active section New Plate Creates a new Plate section below the active section The new Plate section will be a copy of the active Plate section or if no Plate sec tion curr
7. 0 0 e ee eee 6 5 General Information about Enterprise Administrator 6 6 How Enterprise Administrator and the Enterprise Edition of SoftMax Pro Work Together 0 0 cee eee eee eee 6 6 InstallaHODlossnone 2 ag aye Got Gee wees 8 ee Id p 6 7 Administrator Login and Database 6 8 MIG CS PEE 6 14 Ea S 95 425 tt E TEE ETETE EET T ate wad 6 16 Remove q U Ser rir uta rA E A ee ett 6 18 Add SOUWE s 102p 8 xa Ded add IRA OET al ora 6 19 Delete DO Wale ive oer ved ah rt virg r e PE 6 19 Change Passwords wide we dee qe pee edu 6 20 Br prec 6 21 ee a dead a predi med do one edo Ry ee ree 6 23 PRG EAA loei o tote oe RR At SD be cn oR Ge ae str ted sero gc 6 25 LiG6HSe np iia ay ha ean ho weal ag Sie ep ween and eee 6 26 Enterprise Edition of SoftMax Pro 0 00 eee eee eee 6 28 SECUTIEY ICIS s o of ior ar aco tdi tht odere Gee Games eh ao 6 28 BeOtatenien tS ms s dou aie ee Fie ht Uber a dE wr ace eed e teda 6 29 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator Enterprise General Information The Enterprise Edition of SoftMax Pro contains features from both the Life Science and Drug Discovery editions Unlike those editions however the Enterprise Edition links with a separate software package Enterprise Admin istrator This program allows an administrator to create a database in which users electronic signature and privilege information is stored
8. lee 9 29 Chapter 9 File Management and Printing t 9 2 SoftMax Pro User s Manual Chapter 9 File Management and Printing File Creation and Management During routine use of SoftMax Pro you will create open and save numerous files You may wish to create file folders that will make it easy to locate partic ular files It is also important to understand the way in which SoftMax Pro finds and uses protocol files to enable certain assay options automatically Default Protocol SoftMax Pro User s Manual When you install SoftMax Pro certain files and folders directories are created on your computer s hard disk One of these files is known as the Default Protocol so named on the Macintosh called DEFAULT PPR in Windows The Default Protocol file is automatically used as a template for new files you create You can however create one or more different template proto col files When first installed SoftMax Pro looks for the Default Protocol file in the same folder or subdirectory as the SoftMax Pro program file You can instruct SoftMax Pro to look in a different location however through the Assays menu Suppose for example that you create a folder subdirectory called MYASSAYS and that you create a new protocol file called Default Protocol for Macintosh or Default Protocol ppr for Windows within this folder The Assays menu shows two sections at the top a command entitled Set
9. Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 1 B 6 The Display Dialog Box Before or after a plate or cuvette has been read you can click the Disp ay button to open the Display dialog box which allows you to change the way the data is presented Choices available in this dialog box depend on whether a Plate section or CuvetteSet section is currently active which instrument is chosen in the Preferences or is connected to the computer and which read mode you have chosen for example the choice for Plots is not shown if Endpoint mode is selected Figure 7 2 shows the default Display dialog boxes for the different read modes for Plate sections Figure 7 3 shows the default Display dialog boxes for the three possible read modes for CuvetteSet sec tions The Display dialog box is divided into two parts left and right the default display Raw OD is shown to the left of a dividing line the choices for reduced data displays are shown to the right of the dividing line Note data reduction itself is performed separately settings here simply determine how the reduced data will be displayed in the Plate or CuvetteSet section An additional set of choices in the CuvetteSet section Display dialog box determines how an array of cuvettes will be displayed in the CuvetteSet sec tion one cuvette per row three cuvettes per row or in a microplate layout The dialog boxes below show the default display
10. 0 cece cee cee eee 10 64 Data Analysis Summary Formula 10 65 Printa Report ciues ceed anes a Seated aeo dub eee EM 10 67 Chapter 10 Tutorials t 10 2 SoftMax Pro User s Manual Chapter 10 Tutorials Introduction The hands on written tutorials included in this chapter are designed to take you through the basic features of the SoftMax Pro program If you are new to SoftMax Pro we suggest that you read and follow the steps in one of the tutorials before using SoftMax Pro for the first time since it will help you learn to use the basic features of SoftMax Pro quickly To learn more about SoftMax Pro you will want to look through Chapter 3 SoftMax Pro Overview and either Chapter 4 or 5 which describes how to set up for and perform a reading choose the chapter that is appropriate for the instrument you are using We also suggest that you read the introduction to the Formula Reference Guide 04891 If you have not yet installed SoftMax Pro refer to Chapter 2 Instal lation and follow the steps listed there to install the software before proceeding with the tutorial SoftMax Pro User s Manual 10 3 Chapter 10 Tutorials L 3 Tutorial 1 Quantitative Endpoint Assay Tutorial This tutorial section will lead you through the steps required to create and run a quantitative assay utilizing a standard curve This tutorial is designed to be used by both absorbance and fluorescence i
11. PJPVY Q Turn autoread on Delay lo sees Figure 5 2 Instrument Settings Dialog Boxes for Kinetic Mode SoftMax Pro User s Manual 5 7 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Spectrum Mode Wavelengths Fluorescence RFUs j Ex Fixed Em Sweep Em FixediEx Sweep C Luminescence RLUs Excitation Emission Cutoff C Time Resolved RFUs 5o v Start Step Sensitivity t Readings A MCE S Fast Normal Precise Bo Instrument Settings Q 7 PMT Sensitivity Endpoint Well Scan i i Tam esae PEE Fluorescence Wavelengths CARP dere E um 350 i Doo o nn Before First Read E secs m Start Em Stop 750 2 C Luminescence RLLS Em Step 10 Cutoff None C Time Resolved RFUs Sensitivity busy ee reU REMIS Readings 6 PRT Medium Automix i AutoCalibrate Before Off AutoCalibrate IV AutoCalibrate on i Assay Plate Type 96 Vell Standard gt Cancel Wells To Read Read entire plate AutoRead Assay Plate Type Wells To Read Select an assay plate type 96 well plates 96 Well Standard 384 well plates 384 Vell Standard 48 well plates 48 Well Costar 24 well plates 24 Well Costar 12 well plates 12 Well Costar 6 well plates T UD mum cm xm r 6 Well Costar Turn autoread on
12. a STD Standards Concentration vs PTeanValue Z 18e 4 Figure 8 43 Graph with Quadratic Fit 6 56 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual 4 Parameter Logistic and Log Logit If the standard curve has a sigmoidal shape when plotted on the semi log axes it may be appropriate to use either the log logit or 4 parameter fit Both fits are based on the equation E a x B La where D is the Y value corresponding to the asymptote i e the flat part of the curve at high values of the X axis and A is the Y value corresponding to the asymptote at low values of the X axis The coefficient C is the X value corre sponding to the midpoint between A and D The coefficient B describes how rapidly the curve makes its transition from the asymptotes in the center of the curve A large value of B describes a sharper transition Typically B has a magnitude of about 1 Both the log logit and 4 parameter equations are shown on the screen as y A D 14 x C B D O v Zl crape sw Ft 4 Parameter_v Graph concentration y CUR DII CXICIPB J O A B M D Fiti Groupz1 concentration vs Mean salue 0 064 0 779 11 314 0 341 Figure 8 44 Graph with 4 Parameter Fit 6 57 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 8 58 v Z Grap
13. Plate 1 2 3 11 12 13 14 15 2 805 3 105 4 005 4 405 3 405 3 705 4 805 5 305 4 005 4 405 5 805 6 405 UE RSEN 4 805 5 205 7 005 7 705 Calibrate On 7 14 99 j 5 805 6 405 8 505 9 405 PMT Auto 7 59 AM t 4 4 4 Reads Well 6 7 005 7 705 10 305 11 405 X Auto Cutoff s 8 505 9 405 110 305 11 405 12 505 13 805 Wavelength Combination ILm1 T T 1 10 305 11 405 12 505 13 805 15 205 16 805 5 805 6 405 7 005 7 705 8 505 9 405 10 205 11 405 12 505 13 805 15 205 16 805 18 505 20 405 z v Plate 1 B Setup EX Template X Reduction E Disptay in 7 005 7 705 8 505 9 405 10 205 11 405 12 505 12 805 15 205 16 805 18 505 20 405 22 505 24 905 8 505 9 405 10 205 11 405 12 505 12 805 15 205 16 805 18 505 20 405 22 505 24 305 27 505 20 205 aw data in 1000 s Plate 1 1 1 1 4 4 10 305 11 405 12 505 13 805 15 205 16 805 18 505 20 405 22 505 24 905 27 505 30 205 33 505 36 905 2 505 13 805 152051 6 805 18 505 20 405 22 505 24 905 27 505 30 305 33 505 36 905 40 805 45 1 05 15 205 16 8051 8 505 20 405 22 505 24 905 27 505 30 305 33 505 36 905 40 805 45 105 49 805 54 905 18 505 20 405 22 505 24 905 27 505 30 305 33 505 36 905 40 805 45 105 49 805 54 905 60 705 67 005 22 505 24 905 27 505 20 305 33 505 36 905 40 805 45 105 49 805 54 905 60 705 67 005 74 005 81 805 1 3 5 7 9 11 13 15 17 19 21 23
14. Reads wWell 6 X Auto Cutoff avelength Combination Lrn1 Interleaved Display Figure 8 3 Choices for a 384 Well Plate Display Normal Vertical Rotated Large and Interleaved SoftMax Pro User s Manual 8 9 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Endpoint Display Kinetic Display v Plate JA Ir OO m m oO 0 M F 1505 4 1605 4 1705 4 1805 4 Plate 1 6 2005 4 7 2205 4 2405 4 2605 4 2805 4 3105 4 1705 4 1805 4 2005 4 2205 4 2405 4 2605 4 2805 4 3105 4 3405 4 3705 4 2005 4 2205 4 2405 4 2605 4 2805 4 3105 4 3405 4 3705 4 4005 4 4405 4 2405 4 2605 4 2805 4 3105 4 3405 4 3705 4 4005 4 4405 4 4805 4 5305 4 3705 4 a 4005 4 5805 4 6405 4 2805 4 3405 4 3105 4 3405 4 3705 4 4005 4 4405 4 t 4805 4 4405 4 2305 4 4805 4 5805 4 5305 4 6405 4 7005 4 7705 4 4005 4 4405 4 4805 4 5305 4 5805 4 6405 4 7005 4 7705 4 505 4 9405 4 4805 4 5305 4 5805 4 Wavelength Combination Lrn1 v Plate 6405 4 7005 4 7705 4 8505 4 3405 4 10305 11405 aay 177 Endpoint Fluorescence Ex Em Cutoff Lm1 405 450 435 x Auto Cutoff Automix Off Calibrate On PMT Auto Read
15. Standards g ml cking to the Top of the Window Figure 3 6 Example of a Tool Bar Sti SoftMax Pro User s Manual 3 11 Chapter 3 SoftMax Pro Overview 3 12 Opening and Closing Sections Newly created experiments and sections are initially shown in their full size open form You can close them however to show just their tool bars if desired Sections that are created automatically by SoftMax Pro may be shown initially either open or closed depending on the default protocol file in use To open or close a single section within SoftMax Pro click its indicator gt located in the top left corner of the tool bar When an experiment or section is closed the indicator points to the right when open the indicator points down xz Figure 3 7 shows an open experiment containing three sections the first two are closed and the last one is open Standards sgzml Figure 3 7 Closed and Open Sections The Notes 1 and Plate 1 sections are closed the Standards section is open To open or close multiple sections within an experiment or multiple experi ments in a file simultaneously use the Expand and Minimize commands in the View menu Selecting Minimize 3M for Macintosh CTRL M for Windows will close all sections leaving experiments open to show only section title bars Selecting Expand 3 E CTRL E will open all sections NOH If you close or minimize an experiment the t
16. 1 Currently Molecular Devices has tested adapters supplied by both Keyspan and Belkin and found that both work without problems Adapters from other manufacturers may work equally well SoftMax Pro User s Manual 2 3 Chapter 2 Installation xd 8 pin DIN to Connector 8 pin DIN Serial cable Y e _ 1 d EF Ld e Errr a Port 2 auus 8 pin DIN to DB 25 modem cable PvP lee PLD DD DD DDD Figure 2 2 Serial Cables for USB Macintosh Left USB serial adapter Right Top 8 Pin DIN to 8 Pin DIN Serial Cable not the same as a Macintosh printer cable and Right Bottom 8 Pin DIN to DB 25 Modem Serial Cable 1 Plug the USB connector on the serial adapter into the USB port on the back of the Macintosh computer 2 Plug one end of the 8 pin DIN cable into the USB to serial adapter see Figure 2 2 above Plug the other end of the 8 pin DIN cable into the serial port of the MDC Microplate System instrument The USB to serial adapters generally require that you install software for them to function properly After you have installed SoftMax Pro described in a later section access Preferences from the Edit menu Set the serial port to any open port available with your computer typically you will choose the Printer port as most con temporary Macintosh computers have a built in modem that uses the Modem port then choose the appropriate instrument and finally close the dialog box
17. 10 40 SoftMax Pro User s Manual 3 Chapter 10 Tutorials Sensitivity Now check the settings for Sensitivity by clicking that heading on the left side of the dialog box you need to change the default shown on the right side for the number of readings from the default of 6 to our setting of 3 The default setting of High for PMT Sensitivity is fine and does not need to be changed mi Instrument Settings EU x Ol em e vo Endpoint Kinetic Spectrum Well Scan FLEX Options Wavelengths Ex Em Auto Cutoff On 485 525 515 Readings Fast Normal Precise Readings 3 3 PMT High AE A A A omw Ne Timing fe PMT Sensitivity QA eA anseeaseansounsoaassanssenses i Time 200 secs Interval 2 secs Reads 101 Minimum Interval 1 28 secs Automix Before Off Figure 10 45 Instrument Settings Dialog Box Showing Sensitivity Settings Timing Check the settings for Timing by clicking the heading on the left side of the dialog box Enter the following data into the text boxes on the right pane e Run Time secs 200 e nterval secs 2 The last two items will change to show the Number of Reads as 101 and the Minimum Interval as 1 28 secs When you are finished the dialog box should look like the one shown in Figure 10 47 SoftMax Pro User s Manual
18. Pre Read Plate should be set to Off If not click the appropriate checkbox to turn the setting on or off When you are finished the dialog box should look similar to the one shown in Figure 10 3 10 10 SoftMax Pro User s Manual 3 Chapter 10 Tutorials wae Instrument Settings D e Endpoint Kinetic Spectrum Options Automix amp Blanking Automix amp Blanking Lr enn E Cin Before First Read secs STONE MEE Ec ERREUR SE on mBan ep ata ep E suis m ox Read entire plate Nc KJ Q a s Endpoint Kinetic Spectrum Well Scan Options Read Type Fluorescence Wavelengths c Automix A A E AA AA A aep Sepa j Ex Em Auto Cutoff On p spg 485 538 530 Before First Read i secs Sensitivity Readings 6 PMT Auto Before Off AutoCalibrate On Assay Plate Type 96 Vell Standard Wells To Read Read entire plate Figure 10 3 Instrument Settings Dialog Box Showing Automix amp Blanking Absorbance or Automix Fluorescence The Instrument Settings dialog box for some instruments may show additional choices PathCheck AutoCalibrate Assay Plate Type Wells To Read Strips AutoRead etc The online Help index can be used to learn more about these features For example to learn about AutoCalibrate shown in this dialog box choose Index from the Help menu Then type autocalibrate in the first field and double click the result showing
19. Table 4 1 Features Available in the Instrument Settings Dialog Box for Absorbance Instruments Wavelength f Assay Column Pre Read Plate jr i E Timing Strips rnt ien Speed Plate Wavelength AutoRead ung Type Priority Emax Endpoint Vmax TEE Pndpowt Ce e 1 1 ELE SpectraMax 340 250 Plus Tndpoini neu xe a a Te 013 O Spectrum i a a a SpectraMax 190 190 PC PHOBOIRE L ei 1 pL ee Spectrum ew SpectraMax 340PC Endpoint e Je gt gt 9J9f pucr p J p Spectrum ew SpectraMax Plus 4 isa Endpoint ee 2 Lspemum ee E ee Se ee VersaMax Endpoint Kinetic eL 384 Using the SpectraMax Plus only NOH Incubator control is an instrument setup parameter that is not found in the Instrument Settings dialog box SoftMax Pro User s Manual 4 5 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments M Endpoint Mode i etn n Turn pathcheck on DOO zv Plate background constants in OD i Lint bf Note You can pre read the plate instead of using background constants Above SpectraMax 190 190PC 340PC 340PC 8 and VersaMax Plus m Instrument Settings x PathChiteck L Turn pathoheck on RE YRRRRRNRRRRRERERERERIRETEEETENNRRRRRERERERERHRERIRESEESEERN RSERSERSR
20. lt in the Group section Edit Column Selecting a column makes this command active Choosing Edit Column allows you to change the formula associated with the column and to change the column name Mouse Equivalent Double clicking the column performs the same function Button Equivalent fp4 in the Group section Edit Summary Allows you to edit the formula associated with the summary as well as to change the summary name This command is dimmed if a summary has not yet been created First highlight the summary and then select this command Mouse Equivalent Double clicking a summary performs the same function columns to accommodate the largest piece of text in the column Mouse Equivalent Double clicking the line to the right of the column heading performs the same function Hide Hides the selected column from view This command is dimmed if a column has not been selected Mouse Equivalent Dragging the line to the right of the column head ing until it is on top of the line to the left of the column performs the same function Hide Replicates Hides data for all replicates if any from view Show All Shows all columns or replicates that were hidden previously Show Formulas Causes a new row to appear beneath the headings of each col umn showing the formulas used to determine the data in the columns After choosing this command the menu name changes to Hide Formulas Group Settings Opens the Group Se
21. series with all 96 samples highlight all the wells in the plate select the fill direction and set the replicates to 1 Fill from Top one replicate Fill from Bottom one replicate Fill from Left two replicates Fill from Right four replicates Fill from Top three replicates Figure 4 29 Fill Options and Replicate Settings 7 Click OK to save the series and close the dialog box While still in the Template Editor you can view the value assigned to any well by click ing that well The value will be displayed at the top of the Template Ed itor Holding down the Ctrl Shift keys will display the values for all wells Sometimes you may wish the names of a series of samples to incre ment but may not have a concentration or dilution factor that you wish to enter or you want the concentration or dilution factor to remain constant This can be done using the button setting the starting value to 1 or another constant value and either multiply ing by 1 or dividing by 1 In this way the names will increment but the value of concentration or dilution will not change If you don t have a concentration or dilution factor simply deselect the sample descriptor checkbox if it is selected If sample names are set to increment automatically using the Series function be aware that SoftMax Pro will automatically truncate the sample name to three or four characters including the incrementing number if the sample name starts with lette
22. 5 40 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments e Assay Plate Type e Wells To Read e Compound Source e Compound Transfer e Triturate e Pipette Tips Layout e Compound and Tip Columns Settings for FLEX Mode It is essential that the settings for compound sources and dispensing are correct for the operation of the instrument In particular when a plate is selected for a compound source the selected wells must cover an area that is appropriate for the dispensation Also selections for Assay Plate Types Compound Source plates and Pipette Tips Layout must correlate SoftMax Pro will crosscheck these settings when you click OK to close the Instrument Settings dialog box If the settings do not correlate a warning message will be shown and you must correct the settings before you will be allowed to proceed and close the Instrument Set tings The FlexStation performs readings using fluorescence the light emitted by certain substances resulting from the absorption of incident radiation To measure fluorescence accurately it is necessary to reduce light scatter The governing equation for fluorescence is Fluorescence extinction coefficient concentration quantum yield excita tion intensity pathlength emission collection efficiency Two types of scans are possible you can select a fixed wavelength for excita tion and scan emission or vice versa More about this c
23. Chapter 12 SoftMax Pro Remote Command Language Example ReturnStatus lt tab gt lt cr gt Readerlype lt tab gt Version lt cr gt Otate tab Offline Busy or Idle Temperature tab XXX Drawer tab Open or Closed Que tab NumberKemoteCmdelnQueue ReturnTiming This command sends back the number of seconds that it would take to read the current plate For multi plate documents the plate to be used is chosen in the same way as described in the Read command above SaveAs XXX Have SoftMax Pro save the current document file The file will be saved in the current SoftMax Pro Autosave directory which is set in the Preferences dialog accessed through the Edit menu Data files can also be saved to subdirectories of the Autosave directory Notes e SoftMax Pro will append pda to any filename passed in to it e If a file with the same name already exists SoftMax Pro will automatically overwrite the file with NO warning e If the subdirectory does not exist the file will not be saved Examples SaveAs myFile SaveAs mySubDirectory V myFile SelectAll This is equivalent to pressing CTRL A or choosing Select All from the Edit menu This can be used to select data in group tables for copying not needed for copying plate data SelectSect Select a section by name This can be useful for multi plate protocols or for selecting Group tables for copying Examples 12 12 SoftMax Pro User s Manual
24. Message boxes appear to inform you that the action you are attempting needs adjustment in some way Message boxes contain only an button that you can click to remove the message after you have read the information Alerts inform you that the action you are about to take will have conse quences that may not be reversible or may make a significant change within the program Alerts are indicated by an alert symbol Alerts occurring in the Instrument Settings dialog box generally must be corrected before you are able to close the box and begin the assay Other alerts may not prevent an action from continuing but may cause errors after the run is started Alerts in the Instrument Settings SoftMax Pro User s Manual One example of an alert appears when you are defining instrument settings for a Kinetic assay and the read interval is not long enough to allow the required actions to occur The message might look like this Read interwal 15 too short Minimum 3 seconds Figure 11 1 Example of an Alert Message Setting a longer read interval removes the message and allows you to con tinue Chapter 11 Troubleshooting Other examples of alert messages that appear in the Instrument Settings dia log box are found with the FlexStation instrument These are P Possible assay plate overflow Check fluid dispense volumes P Possible assay plate underflow Check fluid dispense walumez P Fluid dispense time is too short Check fl
25. Reported Here Plate or Cuvette with Template Graph Section Plots Created with Initial Data from UE E Information from Microplate d G Tabl Standards Reader R Group Table Reduced Number from Plate Cuvette Reported Here Interpolated Values Results Reported from Graph Section Figure 7 1 Relationship of Sections This chapter discusses the initial display and reduction of data in the Plate section first followed by display and data analysis in the Group Graph and Notes sections 7 3 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 1 B Initial Data Display and Reduction Plate CuvetteSet Section 4 The manner in which data is displayed in a Plate section or CuvetteSet section depends on the type of reading that was performed Endpoint Kinetic or Spectrum but can be customized by choices made in the data display and reduction settings Depending on the type of reading performed display options include e Number or plot displays for raw OD or T with or without reduced number Kesey The number format use of commas or periods in numeric notation chosen for the computer operating system in general is reflected in the way that SoftMax Pro displays data e Number Plot Threshold Ranged or Grayscale with or without reduced number e Display as one or three samples per row or display as microplate layout CuvetteSet section only e SpectraMax Plus 9 or
26. The template should look like the one in Figure 10 9 when you are finished entering the Standards 10 14 SoftMax Pro User s Manual F SoftMax Pro User s Manual Chapter 10 Tutorials m Expernment 1 Plate 1 semple s07 r Series WF Assign Concentration A4 pail Cancel OF Figure 10 9 Template Filled with Blanks and Standards This tutorial example uses seven separate unknown samples with each unknown being placed in four wells The unknown samples should be entered on the template as follows e Sa01 wells A5 through D5 e Sa05 wells E7 through H7 e Sa02 wells A6 through D6 e Sa06 wells A8 through D8 e Sa03 wells E6 through H6 e Sa07 wells E8 through H8 e Sa04 wells A7 through D7 Start by highlighting wells A5 through D5 Select Samples from the Group menu The selected wells will now show the default first sample name for the Samples group Sa01 The template should look like the one below m Experiment 1 Plate 1 Cancel OF Figure 10 10 First Samples Added to the Template 10 15 Chapter 10 Tutorials Continue highlighting the wells as described above assigning new unknown sample descriptors to each subsequent group When all seven unknown samples have been added to the template it should look like this mi Experiment Plateitl Group semear sees Cancel OF Figure 10 11 Samples Added G This tutorial
27. 24 Concentration Concentration Standards jig ml Dragging Right M iii Standards 24 Concentration Concentration Standards ug ml Resulting Column is Wider Figure 10 31 Sizing the Standard Value Column Wider To shrink this column to the minimum size needed to display the complete title click once in the Concentration column to highlight it Then choose Autosize from the Group menu The column will be reduced in width as shown in Figure 10 32 Several columns can be Autosized at one time by highlighting dragging across multiple columns in the Group and then choosing Autosize SoftMax Pro User s Manual 10 27 Chapter 10 Tutorials 10 28 C Figure 10 32 Columns Before and After Autosize Add a Column The columns that are created by default with various Group sections may not always meet all the needs of a particular experiment You can add a column to any Group section and define the purpose of this column in your data analysis By default new columns are added to the right of any selected highlighted column or if no columns are selected after the last col umn in the Group To add a new column to the right side of the Standards Group make sure this section is active Then deselect any highlighted columns by clicking outside the column area Click the Create Column button To or choose Create Column from the Group menu A dialog box will open asking you to enter information for t
28. Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Introduction 1 2 3 4 5 6 7 8 9 10 11 SoftMax Pro User s Manual Reading a microplate or cuvette using SoftMax Pro is a three part process e Prepare SoftMax Pro to collect data e Prepare the microplate s and or cuvette s to be read and e Read the microplate s and or cuvette s Before reading a microplate or cuvette you must create the Plate section s and or CuvetteSet section s and define the instrument settings for the read ing Instrument Settings must be defined prior to collecting data and may NOT be changed after data is collected In addition it is recommended that you define a template and set the reduction and display parameters prior to reading the microplate or cuvette since these parameters determine how data is displayed and analyzed but these last parameters may be set up and or modified after data collection A typical process for preparing collecting and analyzing data is as follows Start SoftMax Pro Create Plate section s and or CuvetteSet section s as needed Define instrument settings use buttons in the Plate section CuvetteSet sec tion tool bars or the Control menu Define the template reduction parameters and display parameters Prepare the microplate s and or cuvette s to be read and place them in the instrument Initiate the reading Save the data file Modify the template
29. Creates new section s or experiment s that are exactly like the active section s or experiment s and positions the duplicated selection s after the currently active section s The names of the new section s or experiment s will have the word Copy ap pended to their name s For example if you duplicate a section named Plate 1 the duplicate section will be called Plate 1Copy Subsequent duplications will append an incremented number after the name e g Plate 1Copy 2 For more information about this command and what is included when a section is duplicated see Du plicate and New on page 9 27 Text Style T CTRL T Allows you to change the attributes of selected text This command is dimmed if no text has been selected Preferences double click the instrument icon Through the Preferences dialog box you can choose settings for the following options e Serial port connection modem port or printer port for the Macin tosh COMI or COM2 for the PC that will be used to connect to the MDC Microplate System instrument A third option Neither should be used when no instrument is connected to the computer e Export format Time versus Plate and whether or not to include labels e The instrument connected to the computer or if none is connected the type of instrument for which the software should be config ured e Whether or not files will be saved automatically as well as
30. If you move the SoftMax Pro window such that the title bar is under the status bar you will have to hide the status bar before you can move the window to see the title bar again EH You may change the size of the window shown in Windows by dragging the T side bottom or corner of the window when the cursor changes into a dou ble headed arrow You can expand the window to the full size of the screen by clicking the Maximize button in the upper right corner of the window If you extend the bottom edge of the window beyond the last section or the right edge beyond the edge of the sections the intervening space will be gray You can also reduce the window to an icon by clicking the Minimize button in the upper right corner of the window When the window is shown as an icon it is still functional double clicking the icon will restore the window to its pre vious size Other options are available for resizing windows refer to the doc umentation that came with your Windows software for more information ZH whose The width of the experiment and sections shown in the window is tiene Ht constrained by the width set for the printed page size The dimen sions and orientation of the printed page and consequently the dis play size may be changed by selecting either Page Setup Macintosh or Print Setup Windows from the File menu 3 6 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Status Dar Near the top
31. Import FLIPR Import brings tab delimited ASCII text file information into SoftMax Pro Export creates a file of Plate section data CuvetteSet section data and or Group table section data All information from SoftMax Pro can be saved as tab delimited ASCII or text format which is suitable for importing into word processing or spreadsheet programs SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview SoftMax Pro User s Manual Import Analyst allows you to import verbose reports generated using Criterion Host or SCREENPLAY software for all instrument modes Import FLIPR allows you to import FPD files exported from FLIPR software For more information on importing or exporting files see Export ing Importing Information on page 9 16 Page Setup Print Setup You should access this command after you select a different printer in the Chooser Macintosh if you choose a different printer using Windows or if you want to use a different paper size or printing option Choosing Page Setup Print Setup opens a dialog box with various options for printing More about this function can be found in the documentation that accompanies your computer and or printer AOH The Page Setup Print Setup command also controls how SoftMax Pro sections are displayed on your monitor the width of the display corresponds to the width of the page size Print Section name Selection Allows you to print just the activ
32. Insert the SoftMax Pro CD into the CD ROM drive Macintosh Double click the CD ROM drive icon to open it Windows Open My Computer and then double click the CD ROM drive icon to open it Double click the Installer icon Macintosh or Setup icon Windows and fol low the screen prompts The Welcome screen suggests that you close all other programs before beginning installation of SoftMax Pro software It is also recommended that you disable any virus protection software you may have running before installing SoftMax Pro Click Cancel to exit the installer and close any pro grams and or disable virus protection and then start the installer again from step 3 above To continue with installation click Next The screen named Choose Destination Location asks you to select the drive where the software will be installed A default location will be shown on the screen If you have more than one hard drive in your computer you may change the destination to the disk drive where you wish SoftMax Pro to reside or accept the default location The Setup Type screen asks you to choose between Complete the default or Basic Choose Basic to install everything except the tutorial files Otherwise click Next to continue SoftMax Pro User s Manual Chapter 2 Installation 7 8 9 OBE The Back button is available on all but the first screen allowing you to go back to previous screens and revie
33. PathCheck on the SpectraMax 190 and 340PC instruments uses only the water constant Therefore samples must be completely aqueous no solvents clear nonturbid and have no interfering contaminants in the wavelength range of 900 to 1000 nm not highly colored in the upper wavelength range as in Lowry assays PathCheck must be chosen in the Instrument Settings dialog box before read ing a plate in order for this feature to be active After reading you can choose whether or not to apply the correction in the Reduction dialog box for that Plate section you can also choose to view the data as percent transmittance 76 T or absorbance OD PathCheck cannot be applied after a reading nor can you change Water Constant to Read Cuvette Reference after a reading doing so will require that you replace the existing data with that from a new reading of both the cuvette and the microplate For more detailed informa tion regarding the calculations used for pathlength correction see Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 4 16 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Automix The Automix function is a patented feature that allows you to set automated shaking of the microplate The options available for Automix depend on the read mode chosen and the type of instrument connected or chosen in the Pref erences SpectraMax and VersaMax Instruments
34. SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Sample names can be 300 characters long however only the first five or six characters will be displayed in the template and in the Plate section You can type a sample name directly into the box next to the arrow for the drop down list by highlighting and typing over the existing sample name If one or more wells were highlighted typing a differ ent name will replace the sample name shown in the template with the name you have typed and if the name is new will add this name to those that can be chosen in the drop down list When wells have been selected in the template and sample names have been defined the sample menu will look like the one below The checkmark indi cates the sample that is currently being named Sarople Staz v Concentration lo mq n Figure 5 25 Sample Drop Down List Sample Names Defined and Wells Selected Sample Descriptor Field The field associated with the sample descriptor only appears in the Template Editor tool bar if the group column type that has been selected requires this type of information Generally the information entered in this field is a dilu tion or concentration Position the cursor in this field and enter a number The label and units assigned to the sample descriptor in the Group Settings dialog box are shown on either side of the field Assign Area The Assign area co
35. The Series function allows you to work with groups of wells in the Template so that the standard value or dilution factor increases or decreases in specified steps The name can be incremented as well SoftMax Pro User s Manual B 13 Appendix B Glossary of Terms B 14 Slope A reduction option that determines the slope of the combined plot i e the slope of the line using linear regression after the wavelength combination reduction This reduction uses all timepoints visible in the reduction win dow Slope is the same as Vmax Rate when Vmax Rate is set to the same num ber of points as the run i e the default Slope is not the same as Vmax Rate if you have modified the number of Vmax Points Spectrum This read mode allows you collect absorbance or fluorescence data across a spectrum of wavelengths This data is displayed as OD RFU or RLU versus wavelength The default reduction displays the wavelength that corresponds to the maximum signal Standard Curve A mathematically idealized representation of the relationship between the concentration of a standard calibrator X value and the OD RFU RLU Y value The standard curve may be set to a linear semi log log log qua dratic 4 parameter logistic log logit 2 parameter logistic exponential cubic spline or point to point curve fits Status Bar Shown above the SoftMax Pro window the Status Bar contains the instrument icon the current temperature within the
36. The limit for the maximum value you wish to report Any values from the reading that are above this limit will not be shown and will be excluded from data reduction The default for MaxOD is 1 0 The MaxOD reduction parameter can be used to exclude the non linear por tion of the reaction from data analysis This type of data reduction is most useful in reactions where the initial portion of the data is linear You might also adjust the End Time setting to remove a non linear portion after a certain time point in the reaction Using MaxOD allows the use of the maximum number of linear points to calculate the slope of the line and thus to deter mine the rate for each well SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Plates 1 Well Graph Reduced 150 Time seca mas Points 13 well B4 L C A DS M max 15 765 az rz5 107 66 R Z p 274 Q 9 ra p 374 150 Time secs Ymax Points 15 well B4 L C6 A DS Ymax 15 765 a a od 101 97 R 0 374 O 9 4 Q 9rr e J Ls Jes JL pene Jr Figure 7 21 Different Reductions of the Same Data Using Different MaxOD Points Both of the Well Graphs shown above display different reductions of the same data these examples use optical density readings In the figure on the top the MaxOD is set to 2 0 for the display of wells B4 C6 and D8 In the figure on the bottom the MaxOD has been reset to 0 5 Alternatively you could leave the Maxim
37. When on you can enter a setting for regulation of the temperature in the microplate chamber Button Equivalent E in the Status bar Calibration Calibration performs an air reference reading at each wave length and updates the non volatile RAM of the instrument thus pro viding new air calibration for the instrument 3 31 Chapter 3 SoftMax Pro Overview 1 F 3 32 Open Tip Drawer FlexStation only Opens the topmost drawer of the FlexS tation instrument that contains pipette tips When this command has been selected and the tip drawer is open the command on the menu changes to show Close Tip Drawer Open Compound Drawer FlexStation only Opens the middle drawer of the FlexStation instrument that contains compounds that can be injected When this command has been selected and the compound drawer is open the command on the menu changes to show Close Compound Drawer You may not open both the tip and compound drawers simultaneously Because of this when you select Open Tip Drawer the Control menu will change to show Close Tip Drawer and the Open Compound Drawer command will be dimmed Likewise when you select Open Compound Drawer the menu will change to show Close Compound Drawer and Open Tip Drawer will be dimmed Set Air Gap FlexStation only This value can be set from 0 to 200 milliliters and sets the distance from the tip of the pipette to the bottom of the mi croplate well The software calculates depe
38. l o IE ooo o Timing Tee Number af Wavelength L BIEN oe Blanking C O Time 5 00 i Pre Read Plate Interval 0 09 Reads 34 j Minimum Interval 0 09 Lint aso v Automix amp Blanking Before Ott Between Ott Pre Read Plate Ott AutoCalibrate Once M P X AutoCalibrate Strips j Read entire plate j Iv AutoCalibrate mm A Cancel Ta nmo 65 m I Figure 4 2 Instrument Settings Dialog Boxes for a Kinetic Reading SoftMax Pro User s Manual 4 7 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Spectrum Mode Wavelengths Start nm Speed Read Stop nm Step nm Automix amp Blanking Endpoint no Atomix M Before First Read SECS Wavelengths a Wavelengths start 350 Stop 750 sausnausunuauscsssaususeuagE EdgFEssunenesssnsnsnsssumunusA SuRAR RR ARRRARRRRRRRRRRRGRRERRARARRRSRRARRRRRRSRSRR RR RR 5 Pre E ead Plate Step 10 em Hm Egencia ee eee ee pom cm acm Pre Read Plate Off Step Sm AutoCalibrate on Strips AutoCalibrate Read entre plate AutoRead i Off Iv AutoCalibrate 2 Off Too nmo 66 oO Y Figure 4 3 Instrument Settings Dialog Boxes for a Spectrum Reading 4 8 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Instrument Settings Opti
39. menu From within the Control Panels choose the Exten sions Manager and turn File Exchange on by placing a checkmark next to it Restart your computer File Exchange will now be enabled 2 Rename the Macintosh files you want to transfer to PC disks so that their names conform to the DOS file naming conventions no more than eight characters no spaces and a three letter extension preceded by a period Table 9 1 below describes the three letter extensions that are associated with data and protocol files from SoftMax and SoftMax Pro Table 9 1 PC File Name Extensions and Macintosh Document Types Tosesf file 3 Letter Macintosh Extension Document eres SoftMax Pro Data File E M SPFd SoftMax Data File SPFd 3 Insert a PC formatted disk into the Macintosh floppy drive It will appear as an icon on the Macintosh desktop SoftMax Pro User s Manual 9 7 Chapter 9 File Management and Printing 1 B 4 Drag the renamed files onto the PC disk icon to copy the files to the disk Drag the disk to the trash to eject it Certain special characters will not translate accurately across plat forms Characters that are created on the Macintosh using the option key such as C or e for example will be shown on the PC as other letters or characters Also if you align items using spaces these may or may not align when viewed on a different platform since fonts on the different platforms have different character widths
40. see Figure 4 12 When enabled this feature can be used to normalize the data acquired from microplate readings to a 1 cm pathlength thereby correcting each OD to the value expected if the sample were read in a 1 cm cuvette achieving signif icantly greater precision Enable or disable pathlength correction by clicking in the checkbox at the top of this section When enabled a checkmark will appear in the box PathCheck PathCheck P L Turn pathoheok Ori mnn etonnnnoisetnntittnanoeeeecontn oO Turn patheheck on menn Pathcheck constant E Flate background constants Cin OD Water constant Cuvette reference Plate background constants Cin OD T You could preread the plate instead of using background constants T You could preread the plate instead of using background constants Figure 4 12 Instrument Settings Showing the PathCheck Option The Instrument Settings dialog box for the SpectraMax Plus or Plus left allows a choice between using a water constant or reading a cuvette reference for PathCheck the Instrument Settings dialog box for the SpectraMax 190 and 340PC right allows only a choice to enable PathCheck because these instruments use a water constant Pathlength correction is used to report OD or percent transmittance for each microplate well with the depth of the fluid in the well normalized to 1 cm Two methods for pathlength correction are available when using the S
41. tors see the accompanying Formula Reference Guide NOt To delete a column highlight it and press the Delete key or choose Cut from the Edit menu 45 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 46 Adding or Editing a Summary in a Group Section To add a summary at the bottom of a Group section click the Create Sum mary button in the tool bar or choose Create Summary from the Group menu The Calculation dialog box will appear allowing you to change the name of the summary or hide the name if desired as well as to enter a description of the summary and to create a formula for it including the number of decimal places for the result if appropriate Double clicking an existing summary causes the same dialog box to appear allowing you to modify summary infor mation Calculation Name Summary O Wide Name Decimal Places Figure 7 34 Calculation Dialog Box Information entered in this dialog box will be placed at the bottom of the Group section as follows Name Description Formula Result You can click the checkbox next to Hide Name which will change the way the summary appears to show Description Formula Result NOJ The name assigned to a summary is important since you can refer ence the summary by name in other formulas building one upon another The summary name can be referenced even if it is hidden If you wish to see the formulas associated with summaries without o
42. typically either modem or printer for the Macintosh and COMI through COM for the PC Serial ports that are not available will be dimmed An additional setting the factory default is Neither This selection is used when you do not have an instrument connected to the computer but wish to use SoftMax Pro or when the port connection must be disabled for other rea sons Choose the appropriate setting for your computer s physical serial port connection and click OK The instrument icon should now appear normally without an X through it m Preferences E E Serial Port COM2 oF Serial Comm Speed 9600 E e Autazawve File gus guns bach TEOTPOTET TU TUO ULES TUS TETTUUTIGTEC TUO TEC TERT TRO ELAR TE Lees EUG TRO ERE HESS TUS TES TESI LEE i MN E Bein C Program FilestsOF Tmax PRO 4 0 E Time Append Date Include Labels oh Ir Append Time mapane weh SUFTmexPRUFle Text File D Add Filename and Date Reader FLEXstation Filters Autoprint Cancel Preferences Serial Port Serial Comm Speed 3600 b ul ET v Autosave File l Export Format E I amp FTmax PRO 4 4 j 2 Time amp Plate j o Append Date Bd Include Labels File Prefix C Append Time O Time Interpolate i amp 5OFImax PRO File t2 Text File j O Add Filename and Date Reader FLEXstation b al Filters Ll Autoprint Figure 2 7 Preferences Dialog Box Windows top
43. wm Display ES Raw Reduced Ymax units per sec Lrm1 Example Options cee eee EE DM EN Display Threshold wr ou Low High Limit Boxes Show wwith reduced number Cancel Figure 7 9 Display Dialog Box for Kinetic Threshold Display Threshold The Threshold display shows the raw data as a plus for values above the high limit an asterisk for values within the limits and a minus for val ues below the low limit Figure 7 10 shows an Endpoint reading displayed using the Threshold option along with the reduced number 7 15 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments v Dj raten IE Plate 1 PURIMBIMMIBM RE EUR 0 111 0122 0135 0 149 0 165 0 182 0 201 0 223 0 246 0 272 0 300 Lm1 450 B 0122 0135 0 149 0165 6 182 0 201 0 222 0 246 0 272 0 300 D zzz 0 367 Calibrate Once 0 142 0165 0 182 0 204 0 223 0 246 0 272 0 300 0 332 0 267 0 406 0 448 a 47 am 7 15 93 Lu D 0182 n 201 0 222 0 246 0 272 0 200 0 332 0 367 0 406 0 448 0 495 0 547 E 0 223 0 246 0 272 0 300 0 332 0 267 0 406 0 448 0 495 0 547 0 605 0 669 F 0 272 0 200 0 332 0 367 0 406 0 448 0 495 0 547 0 605 0 669 0 739 0 817 Ww G 0 332 0 367 0 406 0 448 0 495 0 547 0 605 D 669 0 739 0 817 0 903 0 997 Ww LI H page 0 448 0 495 0 547 0
44. 485 538 530 rMumber of Wavelengths 1 w Sensitivity j Readings 5 Excitation Emission M Auto Cuttoft 2 uds ut fas v Ee Tv 5 Timing Time 300 secs Interval 8 secs Reads 34 Minimum Interval 08 secs Fade tara irc terete Ath eect anat ENDO OMNES Automix Before Off Between Off AutoCalibrate Once Assay Plate Type 96 Well Standard Wells To Read Read entire plate Compound Source Beckman 140504 Figure 5 37 Instrument Settings Dialog Box FLEX This read mode provides access to all of the fluidic capabilities of the FlexSta tion instrument When this read mode is chosen several other choices are enabled in the Instrument Settings dialog box ANNE Molecular Devices recommends that you follow the order of the settings choices from top to bottom when working in FLEX mode since some choices determine the settings that will be available in subsequent choices Following the settings in order is also recom mended to avoid problems that could potentially damage the instru ment Especially important are the settings for the Pipette Tips Layout and Compound amp Tip Columns settings If you change earlier settings after choosing the parameters for either of these make sure to check the settings in these tabs to ensure that the changes are reflected there Choices available within FLEX mode are e Wavelengths e Sensitivity e Timing e Automix e AutoCalibrate
45. 9 11 9 15 11 12 11 13 Clear command 3 28 B 3 Clear button 4 21 4 23 4 28 5 20 5 22 5 26 1 1 Close command 3 26 Close Drawer command 3 31 Column adding deleting 3 28 7 41 7 45 8 38 8 42 8 43 Autosize 3 20 3 38 7 43 8 40 10 26 10 58 copy paste 9 27 Create command 3 20 3 38 editing 3 20 3 38 7 41 7 43 7 44 8 38 8 40 8 41 enlarge 7 42 8 39 format 4 25 5 23 formulas 7 45 8 43 copy paste 9 27 editing 7 43 8 40 modifying 7 44 8 41 showing hiding 3 20 7 41 8 38 hiding 3 38 7 41 7 42 8 38 8 39 reference to other 7 45 8 43 resizing 7 41 7 42 8 38 8 39 showing 3 38 showing hiding formulas 3 20 7 41 8 38 text format 7 47 8 45 Column vs wavelength priority reading 4 44 5 35 Communication instrument icon 2 15 ports Preferences setting 2 16 problems 11 10 serial port 2 16 Connect Points 7 65 8 62 Connections to Macintosh 2 3 2 4 to PC 2 5 Control menu box Windows B 4 Copy 9 25 B 4 command 3 28 9 25 formulas 9 27 Group columns 9 27 Plate data 9 25 pre read data 4 15 template 9 25 Create Column command 3 38 7 45 8 42 Create Summary command 3 36 3 38 7 46 7 47 8 44 8 45 Cross platform files 9 7 Cubic Spline curve fit 7 62 8 59 B 4 Curve fit 7 53 7 54 8 51 8 52 B 4 4 Parameter 7 59 8 57 Cubic Spline 7 62 8 59 B 4 Exponential 7 63 8 60 B 6 judging a good fit 7 64 8 61 Linear 7 55 8 53 B 8 Log Log 7 57 8 55 Log Logit 7 59 8 57 B
46. A dialog box will appear asking you to choose the folder or directory con taining the protocol files you wish to have appear in the Assays menu Select one of the other folders within the SoftMax Pro directory folder or if you have other protocol files in a different location choose that location and then click Select If you do not see any files in the Assays menu or if the files shown are not correct it may be that a previous version of SoftMax Pro was not uninstalled prior to installation of a newer version This can cause SoftMax Pro to default to using the old directory for assay files rather than the directory associated with the most current program version Follow the instructions given above to choose the correct folder for the default assays folder 2 23 Chapter 2 Installation t 2 24 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview CRO CIC DEOS eoe 2o 119 aep aoc db rond os ard oa ears eee owed we 3 3 The SoftMax Pro Window eee 3 3 MIDI Get riuscire iot m cabernet ease aah nM NE ot E 3 6 SLatus Datooue ipee t xen or Bre Sb ovre BE eh Baer ne c 3 7 Experiments and SCCUONSi 2cosads det eR ores Ate SCR ees 3 8 EXDCHINGING 2 dva i seal rE EEEE CEET er es 3 8 DCC OM aca oie bie aa heey ceutical oo anes em o a a Sac ees 3 9 INOLGS SECON 8 Gant ewes ie Ae ep de petas eres 3 13 Plate SECON 29 5 2pm su hn dels mon bean ee ee oe eee 3 15 C uvetteber DECHON eset x oet eR eh ee RENAL T
47. C Pages from 1 to 9999 i 2 jJ E Collate m Options 1 v Experiment Title v Date and Time IV File Name v Page Numbers C Print All Print Report Header T ext caros Figure 10 83 Print Dialog Box for Windows Epson Stylus COLOR 980 shown You have completed this tutorial but SoftMax Pro has many other features that you will find useful The next chapter describes in detail items only briefly discussed up to this point and provides information regarding customizing and using the rest of the features of SoftMax Pro 10 67 Chapter 10 Tutorials t 10 66 SoftMax Pro User s Manual Chapter 11 Troubleshooting Introg t C HEOTLa eon ore Spies aac bed deere Ede pide tocado a extern ae os 11 3 Message Boxes and Alerts 0 eee cece eee eee 11 3 Alerts in the Instrument Settings 22000 11 3 Alerts in Sort Max PLO soit iain Baan tisk tes Be Se ied ent 11 4 Error Messase DORES scdwed Gaudia meetin race eco Sura idees b oda 11 8 Communication Problems 0 0 cece eee eee eee 11 10 Display Problems si vev eye stone E ERREUR Oa eae dedos 11 14 Pantune Problems 44 8 tee este EPI dO dO Euer eats 11 14 General Problems ees 11 15 Chapter 11 Troubleshooting t 11 2 SoftMax Pro User s Manual Chapter 11 Troubleshooting Introduction Alerts message boxes or error boxes that appear while you are using Soft Max Pro usually contain a descripti
48. Chapter 12 SoftMax Pro Remote Command Language SelectSect Plate 0023499 SelectSect Group 1 SelectSectNum Select a section by its order in the document This can be useful for multi plate protocols or for selecting Group tables for copying Example SelectSectNum 3 SetFolderAs XXX Set the current protocol directory You must specify the root directory Example SetFolderAs c SoftMaxPro Basic Protocols SetPort X Tell SoftMax Pro to set a serial COM port Setlemp XX X Send a command to set the current instrument incubator temperature SetUserName Send a command to set the User name prior to opening a protocol file that has been shielded The User name is not the same as the Author name it is the name of the per son using a shielded protocol to gather data The User name is not used to validate an electronic signature for a password protected file Notes e This command tells SoftMax Pro to save the user s name internally so that when an OpenAssay command is issued for a shielded protocol SoftMax Pro will assign this User name for the newly created document e This user name remains stored within the program until the SoftMax Pro is terminated e The user name will be ignored if the currently open data file was not gener ated from a shielded protocol Example SetUserName John Doe SetUserPassword Prepare SoftMax Pro with the protocol password prior to opening
49. CuvetteSet Section Tool Bar Data Display Reduction Settings Instrument Setting SoftMax Pro User s Manual lt gt fl cuvetteset 1 a E Template EZ za CuvetteSet Ai o 1 Ya es Endpoint 0 011 00 0 000 Ob 0 012 Ob Sint fred Data 6 30 99 3 35 PM Data 6 30 99 3 46 PM Data 6 20 99 3 46 PM Ref 6 30 99 3 35 PM Ref 6 30 99 2 35 PM Ref 6 30 99 3 35 PM Wavelength Combination Lrat Data Mode Absorbance Figure 3 12 CuvetteSet Section The CuvetteSet section allows you to collect data from the cuvette port of the SpectraMax Plus or Plus define a template to be used for analysis define how the data will be displayed and define the type of data reduction The Template Editor in the CuvetteSet section is used to describe the contents of the Cuvette Choosing a particular function or command in the CuvetteSet section can be done in several ways You can use the mouse to click on a button choose a command from one of the drop down menus in the menu bar or press a key stroke combination to activate a command or function The CuvetteSet section is divided into four areas e Tool bar e Data display can be shown in three different ways three samples per row one sample per row and in a grid of cells which is similar to a microplate format e Instrument settings shown to the right of the data display e Reduction settings shown at the bottom of the data display When colo
50. Edit Template Figure 6 2 Template Page SoftMax Pro User s Manual 6 9 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator Defining templates can save time when assigning privileges especially when the number of users is large Templates are sets of privileges that can be assigned quickly Add New Template Clicking this button opens a dialog box asking that you name the template provide a description of it if desired and then choose the privileges that will be associated with that template Delete Template After selecting a Template from the list clicking this button asks you to confirm that you wish to delete the template Choosing will remove the template from the database Edit Template Clicking this button allows you to make changes to the privi leges associated with the selected template Modify User a X User Privileges for Charles Lexington Registered software SoftMax Pro Save files and protocols Sign e statements Create and modify e statements Clear Audit Trail Change Autosave parameters Change Group Table and Template settings Change Data Reduction Settings Change Instrument Settings Add New Software Delete Software Password Change the password of current user Change Password Force Password Change cea Figure 6 3 User Privileges Page in Add New User Wizard The Registered software drop down box shows available software programs fr
51. Ig I where I the light intensity after it passes through the sample Ip initial light intensity generally light traveling through air When reading a microplate there are two ways in which the MDC Microplate System instrument can obtain an Ig value with the air reference information programmed into the NVRAM non volatile memory of the instrument or through instrument autocalibration a reading of an air reference prior to reading samples In early versions of SoftMax and SoftMax Pro a selection was available in the Instrument Settings dialog box to turn autocalibration on 4 39 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments k 4 40 or off In SoftMax Pro version 2 4 and above but prior to version 3 0 autocali bration could not be enabled or disabled by the user Instead the software made the determination regarding whether or not to autocalibrate Version 3 0 again made it possible to choose whether or not autocalibration will occur via a choice on the Instrument Settings dialog box See page 4 18 for more information When reading a cuvette the SpectraMax Plus or Plus uses the REF value typically a reading on the appropriate buffer as Ip If no reference reading is taken the instrument uses the air reference information programmed into the NVRAM of the instrument Reading a Microplate You can start a reading at any time after defining instrument settings It is not necessary
52. Lm1 Pathlength and Custom for all MDC Microplate System instruments when you read at a sin gle wavelength or if you choose 1 Ref for all instruments except Spectra Max and VersaMax The Pathlength reduction reports the pathlength in each well With all SpectraMax instruments additional wavelength combinations become available depending on the number of wavelengths chosen For any MDC Microplate System instrument you can choose Custom and create dif ferent reduction formulas The wavelength combinations available in the drop down menu show the correct format of the formulas that produce these reductions PathCheck SpectraMax Plus Plus 9 190 190PC 340PC and 340PC only SoftMax Pro User s Manual Pathlength correction allows microplate data in Endpoint mode for those instruments that support PathCheck to be normalized to a 1 cm pathlength When PathCheck is turned on in the Instrument Settings dialog box for the plate Apply PathCheck appears in the associated Reduction dialog box l The Pathlength option is only applicable to Plate or CuvetteSet sections when using the SpectraMax 190 190PC 340PC 340PC3 Plus or Plus 64 however the wavelength reduction display is not instrument dependent in Soft Max Pro so this choice is always shown regardless of which instrument is connected or chosen in the Preferences Choosing Pathlength with any other instrument or for CuvetteSet sections in the SpectraMax
53. Macintosh bottom SoftMax Pro User s Manual 2 15 Chapter 2 Installation Set Preferences Serial Port 2 16 If the port setting is correct and the MDC Microplate System instrument is on yet communication is still not functioning properly quit SoftMax Pro turn off power to both the computer and the MDC Microplate System instrument and check that the cable connections between the instrument and computer are secure Turn both machines on again and restart SoftMax Pro If an X still appears over the icon refer to Chapter 11 Troubleshooting for more infor mation It is a good idea to set the SoftMax Pro preferences before proceeding to use the program The preferences include certain configuration settings such as which port is used for the connection to the instrument filters for Emax Vmax UVmax and ThermoMax as well as settings for automatic printing and saving of files and the format for exported data To make these selec tions choose Preferences from the Edit menu to bring up the Preferences dia log box see Figure 2 7 Check that the setting for the serial port matches the physical port used to connect the computer and the instrument The example dialog box shown at the top of Figure 2 7 shows the serial port set to the Macintosh modem port The Windows dialog box example shows the serial port set to None Mane CO W CON COT CO COT i ey COE COT EON Figure 2 8 Serial Port Selection in
54. Plate MeNi PP 3 33 CuvetteSet Menu i e eR REOR ERR renerne 3 35 INOICS MICU P thon esto near nee ane E nes 3 36 Crap MONI a2 ca2cen cee ccd onsen asada sae iav 3 37 COUP VICHY on nea dure qoae oP ebur qom ps d ae oe eae 3 38 Window Menu 2o us 9 3 62 143 1 Orr bdo pad PR on dios dod 3 39 Help Menu Windows eee nnne 3 39 Help Menu Macintosh 0 00 eee eee eee eee 3 39 IrnterprocesedVIe5s dE ITE scie aea delit ken daleey bebe aed oo ae esa 3 40 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instrum n S 4 64 ne ones ES T4 priri EE T ded da NE QR S sears tees 4 1 tbi OQ DCHOTLS o ou cea donis Our OR ER tease Pec E UP MOOR aye ene ad iu ed 4 3 Merum at DOE Uie Baeuucos s dabo sunm P E Edo P ho eang Rm d Sud d 4 4 Instrument Settings Options 0 cee eee eee 4 9 Template BOE Sa cuoxos 9t arabe a ded veu sa E E pases es 4 20 Selecting Wells or Cuvettes in the Template Editor 4 22 The Template Editor Tool Bar 0 00 eee ee eee 4 23 DIUINO La csse eas det ens viera eem Geese mad eq E 4 33 Copying and Pasting Templates 00 4 35 Exporting and Importing Templates 4 37 Reading a Microplate or a Cuvette 0 0 0 0 e eee 4 39 Reading d IMUCIODIAG o eid erred or yal aba ree pas 4 40 Reading a Cuvette SpectraMax Plus and Plus384 only 4 4 General Information about a Reading 4 43 SoftMax
55. SPECTRAmax 34UPC 384 SPECTRAmax GEMINI SPECTRAmax GEMINI 5 v FLEXstation Figure 2 11 Reader selection in Preferences dialog box If the computer is not connected to an instrument the Reader type defaults to the last reader connected but you can choose a different instrument from the menu It is not required that you choose a particular MDC Microplate Sys tem instrument prior to writing a protocol using SoftMax Pro when the computer is not connected to an instrument Choosing an instru ment first can be useful however since the options available in the Instrument Setup dialog box depend on the choice of instrument for example with a SpectraMax you can choose up to six wavelengths but you cannot do this with a Vmax The use of this button applies only to Emax Vmax UVmax and ThermoMax instruments this button is dimmed when another instrument is selected SoftMax Pro User s Manual Chapter 2 Installation Clicking the button brings up a dialog box showing six filter posi tions Filters Lancel Figure 2 12 Filters Dialog Box N KANONA Check that the filter wavelength shown for each position accurately reflects the wavelength of the filter installed in each posi tion of the filter wheel Highlight and change type over incorrect values If the settings in this dialog box are not correct subsequent readings will generate incorrect data While the SpectraMax 250 can also accept two optional fil
56. The Macintosh computer has You can purchase a serial switch that will allow you to choose between only one serial port and can t communication with the MDC Microplate System instrument or the connect to a MDC Microplate printer See Communication 3 below for more information System instrument and printer at the same time Communication 1 11 10 Instrument Icon Shown with an X If the instrument icon shown in the upper left corner of the SoftMax Pro win dow has an X through it check whether the MDC Microplate System instrument is turned on If it is then check the SoftMax Pro Preferences set tings see Setting Preferences below to ensure that the proper communica tions port modem or printer for the Macintosh COMI or COM2 typically for the PC is selected This setting should agree with the physical port to which the cable from the instrument is connected to the computer If the port SoftMax Pro User s Manual Chapter 11 Troubleshooting Communication 7 2 Communication 3 SoftMax Pro User s Manual setting is correct the instrument is on and communication is still not func tioning properly quit SoftMax Pro turn off power to both the computer and the MDC Microplate System instrument and check that the cable connections between the instrument and computer are secure Turn both machines on again and restart SoftMax Pro If an X still appears over the icon it may be that you are using the wrong
57. This curve fitting option fits a linear equation to each pair of data points Port A connection or socket on the computer Ports are common used for connect ing devices such as printers or monitors to your computer and for sending information to and or from your computer to such devices SoftMax Pro User s Manual B 11 Appendix B Glossary of Terms B 12 Protocol File Contains the template s instrument settings and reduction parameters Pro tocol files are useful if you repeat a particular type of assay frequently Quadratic Curve Fit A mathematical description of a parabola y A Bx Cx RAM Random Access Memory The memory that can be used by applications to perform necessary tasks while the computer is on When you turn the computer off all information in RAM is lost Ranged Data Display Raw or reduced data is assigned proportionally to integer values from 0 through 9 based on user defined limits Values above the high limit are dis played as plus and values below the low limit are displayed as minus Raw Data Signal reported from the MDC Microplate System instrument with no alter ation This will be reported as OD RFU or RLU depending on the instru ment type Read Interval The interval of time between the start of one reading and the start of the next reading during a Kinetic or FLEX run Read Button or Command Starts the reading process and shows the Endpoint Kinetic Spec
58. Title Bar Menu Bar Minimize Button Maximize Y Button m 5UFTmax Pro File Edit View Control Assays Window Help 21 a fe Ee PW Untitled TIAL xt Close x ce Experiment 24 a box uy Motes B fal ia Scroll Arrow Platea eg Setup E Template Reduction Ez Display Z a T Autamix Ott T j o o lo Cabton 00 ee ee Bar E a Instrument Settings a OCE ECE Box 5 Wavelength Combination Lrnt Data Mode Absorbance Ver tical m GJ cuvetteseta1 E Setup E Template E Reduction Ez Display ke zz E Scroll Bar CuvetteSet 1 Endpoint Lind 450 Data Mo Data Ref Mo Reference Wavelength Combination Lrnt Data Mode Absorbance Figure 3 3 SoftMax Pro Screen and Window for the PC Windows SoftMax Pro User s Manual 3 5 Chapter 3 SoftMax Pro Overview Window Size Youmay change the size of the window shown on the Macintosh by dragging the size box at the bottom right corner of the window in any direction You can expand the window to the full size of the screen by clicking the zoom box in the upper right corner of the window If you extend the bottom edge of the window beyond the last section or the right edge beyond the edge of the sections the intervening space will be gray To resize the window again drag the size box to a different position or click the zoom box again Clicking the zoom box will restore the previous window size
59. Transferring PC Files to a Macintosh Third party software is available for the Windows compatible PC that allows the insertion and reading of Macintosh 3 1 2 inch diskettes if your Macintosh is equipped with a floppy disk drive If you need to transfer files between Macintosh and PC platforms and do not have such software for the Windows compatible PC you can use PC diskettes or Zip disks for both plat forms since the Macintosh will be able to read them Macintosh computers are equipped with software that allows you to read and transfer files from PC disks PC files can be opened directly within Soft Max Pro as follows 1 Choose Open from the File menu The dialog box that appears has a drop down menu to select the file type The last item on this menu All Documents allows you to see all text documents with standard SoftMax Pro for Windows extensions When selected the file is first given a MacOS look and feel and is then opened in SoftMax Pro 2 Repeatsteps3 through 5 for other PC file name extensions with which you will be working Saving Files Manually 9 8 SoftMax Pro provides several ways to save files manually If the current file has not been saved is Untitled it can be saved under any name If you have opened and modified an existing file you can save it under the same name overwriting the old information or under a different name You can also save the file in the same or in a different location on your
60. but can be customized by choices made in the data display reduc tion settings and the Edit menu e Resizing hiding columns e Adding deleting columns e Showing editing column formulas e Adding editing summaries e Changing text style Default displays for Group sections are shown automatically unless you choose a different display option The particular font to be used in a Group section table as well as its size and style can be changed by first selecting one or more columns in the Group sec tion table and then choosing the Text command from the Edit menu and mak ing the desired choices from the dialog box that appears Settings from the Plate Section Affect Data Display in Group Sec tion Tables Reduction options in the Plate section allow you to display the raw data received from the FlexStation instrument in a reduced analyzed form Which reduction options are available depend on the read mode used and SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments reduction settings chosen Reduced number from the Plate section is dis played in the Values column of the Group section tables by default Reduction settings do not affect the raw data but simply cause it to be dis played differently You can change reduction settings without affecting the raw data from the plate reading in any way Custom reduction formulas require the use of accessors and ope
61. i For this example the user hae eet up a menu selection to soliciting instrument status from SOFTMaxPro application software ld IDM_COPYDATA defined in resource case IDM_COPYDATA sendCommand void sendCommand 1 User hae selected menu selection to send SoftMaxFro remote command ReturnStatus See SoftMaxFro User s Manual chapter 12 Remote Command Language char cmdStr ReturnStatus char msgStr new char strlen cmdStr 1 Place command in heapepace Note that a run time error may occur unless command ie copied to heapspace etrcpy meg9tr cmdStr This struct ie defined in MFC include files COFYDATASTRUCT ca SoftMax Pro User s Manual 12 17 Chapter 12 SoftMax Pro Remote Command Language 12 18 Handle to a window type HWND cd dwData DWORD hWnd Length of message string plus 1 cd cbData strlen msgStr 1 Remote commana string ie in heapspace ca lpData megotr Primary method in sending windows command to SoftMaxFro application HWND hwnd FindWindow SOFTMaxProMainWna SoftMax Pro UINT smaxMgg RegisterWindowMessage SOFTMaxFroMsg SendMessage hwnd WM_COPYDATA WPARAM smaxMsg LPARAM amp cd Message sent delete heapepace no longer used delete msgStr SoftMax Pro User s Manual Appendix A SoftMax Pro Version Specifications IntroQ uu coti erie or duoi ooo PUE I ER dee See dE ei
62. 1 Well Graph Reduced ILm1 20 40 60 80 100 120 140 150 180 200 220 240 260 280 300 Time secs Vmax Points 14 Vell o B4 n D6 A F8 Ymax Per Second 14 267 26 192 45 222 0 970 0 967 D 974 R 2 Figure 8 11 Well Graph for Three Kinetic Wells of a Microplate Raw Data The choices you make for viewing the data in the Plate section also affect the way the Well Graph appears Figures 8 10 and 8 11 show Well Graphs for raw data displays If you display one of the reduced options reduced number Threshold Ranged or Gray Scale the Well Graph will show the plot with an indicator of the reduced value and will include the word Reduced in the window title Zoom Box You can enlarge the Well Graph even further by clicking the zoom box in the upper right hand corner of the window the center box of three in the upper right corner in Windows Clicking the zoom box once will cause the window to fill the total display on your monitor clicking the zoom box again will return the window to its previous size Size Box Clicking and dragging the size box located in the lower right corner Macintosh or clicking and dragging the lower right corner of the window Windows allows you to make the Well Graph any size desired Printing the Well Graph You can print the Well Graph window if desired by clicking the Print button at the bottom left corner of the dialog box If you enlarge the Well Graph to greater than 12 inches the gr
63. 10 Instrument Settings Dialog Box Showing Automix amp Blanking Absorbance or Automix Fluorescence 10 11 Template with Plate Blank Wells Assigned 00 00 cee e eee eee 10 12 Template with First Standards Entered 0 c eee eee eee eee eee 10 12 SoftMax Pro User s Manual SoftMax Pro User s Manual Figure 10 6 Figure 10 7 Figure 10 8 Figure 10 9 Figure 10 10 Figure 10 11 Figure 10 12 Figure 10 13 Figure 10 14 Figure 10 15 Figure 10 16 Figure 10 17 Figure 10 18 Figure 10 19 Figure 10 20 Figure 10 21 Figure 10 22 Figure 10 23 Figure 10 24 Figure 10 25 Figure 10 26 Figure 10 27 Figure 10 28 Figure 10 29 Figure 10 30 Figure 10 31 Figure 10 32 Figure 10 33 Figure 10 34 Figure 10 35 Figure 10 36 Figure 10 37 Figure 10 38 Figure 10 39 Figure 10 40 Figure 10 41 Figure 10 42 Figure 10 43 Figure 10 44 Figure 10 45 Figure 10 46 Figure 10 47 Figure 10 48 Figure 10 49 Figure 10 50 Figure 10 51 Figure 10 52 Figure 10 53 Figure 10 54 Figure 10 55 Template showing First Standards Concentrations 0ee eee 10 13 ee D e DOM 10 14 Template with CTRL Shift Keys Depressed 2 00 cece eee ee 10 14 Template Filled with Blanks and Standards 00 e scene eee ee 10 15 First Samples Added to the Template 0 c eee eee eee eee 10 15 DAM PICS ROCCO
64. 10 41 Chapter 10 Tutorials m Instrument Settings i x Qi 4j e Endpoint Kinetic Spectrum Well Scan FLEX Options Wavelengths Ex Em Auto Cutoff On 485 525 515 i Sensitivity Run Time secs E S Interval secs i Number of Reads 101 Time 200 secs Minimum Interval 1 28 secs Interval 2 secs Reads 101 Minimum Interval 1 28 secs 0 Automix Before Off Between Off AutoCalibrate Once Assay Plate Type 96 Well Standard clrbtm Wells To Read Read area 41 H10 Compound Source Beckman 96 2 3mL v Cancel Figure 10 46 Instrument Settings Dialog Box Showing Timing Automix Check the settings for Automix by clicking the heading on the left side of the dialog box on the right Automix Before should be set to Off Between Reads should also be Off If not click the appropriate checkbox to turn the setting on or off When you are finished the dialog box should look like the one shown in Figure 10 47 m Instrument Settings x Endpoint Kinetic Spectrum Well Scan FLEX Options Wavelengths Ex Em Auto Cutoff On 485 525 515 Automix sonani i Before First Read secs Readings 3 i i PMT High Between Reads secs X d Timing Time 200 secs Interval 2 secs Reads 101 Minimum Interval 1 28 secs Before Off Between Off AutoCalibrate Once Assay Plate Type 96 W
65. 15 205 16 805 18 505 20 405 7 005 7 705 8 505 9 405 10 3051 1 405 12 505 13 805 15 205 16 805 18 505 20 405 22 505 24 905 8 505 9 405 hozosh 1 405 12 505 13 805 1 5 205 16 805 18 505 20 405 22 505 24 905 27 505 30 305 10 305 11 405 12 505 13 805 15 205 16 805 18 505 20 405 22 505 24 905 27 505 30 305 33 505 36 905 12 505 13 805 15 205 16 805 18 505 20 405 22 505 24 905 27 505 30 305 33 505 36 905 40 805 45 105 15 205 16 8051 8 505 20 405 22 505 24 905 27 505 30 305 33 505 56 905 40 805 45 1 05 42 805 34 905 18 505 20 405 22 505 24 905 27 505 30 305 23 505 36 905 40 805 45 105 49 805 54 905 60 705 67 005 22 505 24 905 27 505 20 305 33 505 36 905 40 805 45 105 49 805 54 905 60 705 67 005 74 005 81 805 Lm1 Automix Off Last Read Ex 405 Calibrate On 7 16 99 Em 450 PMT Auto 7 44 AM Cutoff 435 Reads Well 6 Fluorescence Auto Cutoff Wavelength Combination Lrn1 Large Display paper size set to letter Juorescence Reads Well 6 x Auto Cutoff avelength Combination ILm1 Interleaved Display Figure 7 4 Plate Section for the SpectraMax Gemini or Gemini XS Choices for a 384 well plate display are shown Normal Vertical Rotated Large and Interleaved 7 10 SoftMax Pro User s Manual Chapter 7 Displaying and
66. 2 4 6 8 10 12 14 16 18 20 22 24 41 1 61 1 81 2 21 2 61 3 11 3 71 4 41 5 31 6 41 7 71 81 221 2 61 3 11 3 71 4 41 5 31 5 41 7 71 9 41 11 4 151 3 11 3 71 4 41 5 31 6 41 7 71 9 41 11 4 13 8 16 8 121 4 41 5 31 6 41 7 71 9 41 11 4 13 8 16 8 20 424 1 31 6 41 7 71 9 41 11 4 13 8 16 8 20 4 24 9 30 3 36 9 45 1 5 81 71 9 41 11 4 13 8 16 8 20 4 24 9 30 3 36 9 45 1 54 9 1 4 13 8 16 8 20 4 24 9 30 3 36 9 45 1 54 9 67 0 81 8 5 8 20 4 24 3 30 3 36 9 45 1 54 3 67 0 81 8 99 8 122 51 1 81 2 21 2 61 3 11 3 71 4 41 5 31 6 41 7 71 9 41 21 2 61 3 11 3 71 4 41 5 31 6 41 7 71 9 41 11 4 13 8 111 3 71 4 41 5 31 6 41 7 71 9 41 11 4 13 8 16 8 20 4 I 41 5 31 6 41 7 71 9 41 11 4 13 8 16 8 20 4 24 9 30 3 36 9 4 81 1 41 7 71 9 41 11 4 13 8 16 8 20 4 24 9 30 3 36 9 45 1 1 41 11 4 13 8 16 8 20 4 24 9 30 3 36 9 45 1 54 9 67 0 3 8 16 8 20 4 24 9 30 3 36 9 45 1 54 9 67 0 81 8 99 8 10 4 24 9 30 3 36 9 45 1 54 9 67 0 81 8 99 8 122 149 indpoint Lmi Automix Off Last Read 405 Calibrate On 7 16 99 450 PMT Auto 7 44 4M Lm1 Automix Off Last Read Ex 405 Calibrate On 7 16 99 Em 450 PMT Auto 7 44 AM Cutoff 435 peads Well 6 Auto Cutoff Wavelength Combination Lrn1 Fluorescence Large Display paper size set to letter luorescence
67. 21 5 20 import export 4 37 9 24 samples 4 21 5 20 selecting wells 4 22 5 21 series 4 30 5 27 fill direction 4 31 5 29 Template Editor 3 33 3 35 4 20 4 22 5 19 5 21 5 51 10 12 10 48 Group area 4 23 5 22 tool bar 4 23 5 21 Text format 9 14 Text Style command 3 29 9 14 Threshold display 7 15 8 11 Time at 1 2 Maximum 7 35 8 28 8 32 B 14 Time at Maximum 7 35 8 28 8 32 B 15 Y Time at Minimum 7 35 8 28 8 32 B 15 l Time to Vmax 4 10 5 11 7 34 8 27 8 32 Y axis command 3 37 7 67 8 64 Time resolved fluorescence B 15 Title Bar B 15 Z Title screen 2 13 2 14 3 3 Zero Baseline 8 34 Tool bar 3 11 Zoom box 7 21 8 17 Transmittance T 1 4 4 9 4 11 4 16 7 12 7 18 7 24 7 36 7 37 9 20 B 5 B 13 B 15 Troubleshooting Technical Support 1 12 U Undo command 3 28 B 15 Uninstall 2 9 Units group 10 12 User name B 15 Users Page 6 10 V Values absolute 7 18 7 31 7 32 8 24 8 25 8 28 8 34 Vertical Display B 16 View menu 3 30 Vmax 4 10 5 11 7 33 8 26 8 31 Vmax kinetic reduction 7 33 8 26 8 31 Vmax Points 7 18 7 29 7 30 7 33 7 34 8 15 8 22 8 23 8 26 8 27 8 31 8 32 W Wavelengths 4 11 Spectrum 4 13 5 15 Well graph 7 18 7 21 8 14 8 17 printing 7 21 8 17 9 11 Well Scan 1 4 Wells graphing 7 18 8 14 masking 7 22 8 18 selecting in template 4 22 5 21 Wells to Read 5 18 5 43 Window active 3 26 3 34 B 1 Page Setup command 3 6 scroll bar 3 11 s
68. 33 455 S Plate E Template E Display 15 1 39 6 755 1 8 455 20 355 22 455 24 855 27 455 30 255 53 455 56 855 40 755 18 455 20 355 22 455 24 855 27 455 30 255 3223 455 36 855 40 755 45 055 43 755 Raw data in 1000 s Plate 1 l H 22 455 24 855 27 455 30 255 33 455 26 855 40 755 45 055 49 755 54 855 60 655 3 955 4 355 4 755 5 255 5 755 6 355 6 955 7 655 8 455 9 355 4 755 5 255 5 755 6 355 6 955 7 655 8 455 9 355 10 255 1 1 355 4 4 5 755 6 355 6 955 7 655 8 455 9 355 10 255 11 355 12 455 13 755 6 955 7 655 s455 m 10 255 11 355 12 455 13 755 15 155 16 755 8 455 9 355 10 255 11 355 12 455 13 755 15 155 16 755 18 455 20 355 10 255 1 1 355 12 455 13 755 195 155 16 755 18 455 20 355 22 455 24 855 12 4935 13 755 15 155 16 755 18 455 20 355 22 455 24 855 27 455 30 255 I OO mo m oO 0 M rr 15 195 16 755 18 45520 355 22 455 24 855 27 455 30 255 32 455 36 855 19 455 20 2539 22 455 24 8523 27 495 30 255 33 452 36 855 40 753 45 093 Vertical Display TU Raw data in 1000 s Plate 1 2 3 11 12 13 14 15 2 805 3 105 4 005 4 405 3 405 3 705 4 805 5 305 4 005 4 405 5 805 6 405 4 805 5 305 7 005 7 705 5 805 6 405 8 505 9 405 Lm1 Automix Of
69. 340PC only If you have chosen 384 well plates from the Instrument Settings you can display the wells in different ways Normal Vertical Rotated Large or Interleaved Default displays are shown automatically unless you choose a different dis play option Reduction options allow you to display the raw data received from the MDC Microplate System instrument in a reduced analyzed form Which reduction options are available depend on the read mode instrument and read type used custom settings using formulas you create are available for all read modes instruments and read types Reduction settings do not affect the raw data but simply cause it to be dis played differently allowing you to change reduction settings without affect ing the raw data from the plate or cuvette reading in any way Custom reduction formulas require the use of accessors and operators that are understood by SoftMax Pro A list of such accessors and operators can be found in The Formula Reference Guide SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments The Data Display SoftMax Pro User s Manual After a plate or cuvette has been read the information sent from the MDC Microplate System instrument is shown in the data display in the active Plate or CuvetteSet section The default display for data from Endpoint readings is raw OD numeric values Data from Kinetic or Spectrum readings is displayed in
70. 5 51 Figure 6 1 Figure 6 2 Figure 6 3 Figure 6 4 Figure 6 5 Figure 6 6 Figure 6 7 Figure 6 8 Figure 6 9 Figure 6 10 Figure 6 11 Figure 6 12 Figure 6 13 Figure 6 14 Figure 6 15 Wells to Read Dialog Box with Wells A3 G7 Selected Luesue 5 19 Template Editor Dialog BOX erint mr RUE owe qe Re Eee ba prae s 5 20 The Appearance of the Template Editor Tool Bar under Different Circumstances uou eurer zr Pa anr rhet e A e Ma Oe E Ropa 5 21 The Group Drop Down List eeeeeeeeeeeeee ee 5 22 Group Settings Dialog Box Using Default Settings 0 0 5 23 Group Settings Dialog Box for the Group Named Patient 1 5 25 Groups Settings Dialog Box Showing Original Column Format 5 25 Modifying Sample Names and Descriptor Values in the Template Editor 5 26 The Sample Drop Down List 0 cece eee eee eee 5 26 Sample Drop Down List Sample Names Defined and Wells Selected 5 27 Assign Area of the Template Editor Tool Bar 0 00 e eese 5 27 Template with Wells Selected for a Series 00 cee eee eee eens 5 28 Dees DIG OS DOR EP rnm 5 28 Fill Options and ReplicateSettings leeren 5 30 Graphic Representation of Plate Blank Subtraction 02 0005 5 31 Template Editor Showing Plate Blank and Group Associated Blank Wells 5 32 Copy Template and Paste Template Commands in the Plate Me
71. 9 minimum number of standards 7 64 8 61 Point to Point 7 61 8 59 B 11 Quadratic 7 58 8 56 B 12 Semi Log 7 55 7 56 8 53 8 54 B 13 Custom formulas 7 67 8 64 reduction 7 38 8 35 Cut command 3 28 B 5 Cutoffs 5 15 B 4 CuvetteSet menu commands buttons 3 17 3 35 reduction options 7 4 8 4 resizing the display 7 11 7 12 section 3 17 7 4 B 4 data display 7 4 Settings command 3 36 D Data exporting importing 9 16 9 21 Data display 4 45 5 36 5 52 7 3 7 4 7 5 7 41 8 3 8 4 8 5 8 38 10 22 10 54 B 5 Gray Scale 7 15 7 17 8 11 8 14 Group section 7 41 8 38 limits 7 15 8 11 Ranged 7 15 7 16 8 11 8 12 reduced number 7 14 8 8 Threshold 7 15 8 11 well graph 4 45 5 36 5 52 7 18 8 14 Data file 2 9 2 10 7 3 8 3 9 5 B 5 locating 9 5 ON z UM N Con Us N m Data points 7 30 7 31 7 64 7 8 61 8 64 11 14 one or more points lost 11 6 Data reduction 1 3 7 23 8 19 calculation hierarchical 7 23 8 19 Database Enterprise Administrator 6 8 Default protocol 9 3 B 5 Delete command 3 9 3 10 4 21 5 20 Delete Section command 3 28 Desktop B 5 Dialog box B 5 Dilution factor B 5 Dimmed B 5 SoftMax Pro User s Manual Display command 3 33 3 35 default settings 7 6 7 12 8 6 8 8 dialog box 7 6 8 6 options 9 17 parameters 7 3 8 3 problems 11 14 reduced number 7 14 8 8 Drawer button 3 8 Dual wavelength 7 39 Duplicate command 9 27 B 6 E Edit Column command 3 38 E
72. A 3 SoftMax Pro 3 1 Update Specification 0 0 0 0 0006 A 3 SpectraMax Plus384 support 0 0 0 0 eee eee A 3 SpectraMax Gemini XS support 0 0 0 0 cece eee A 3 Well Scanning Read Mode 0 0 cece eee eee A 3 Apple OS 9 Navigation Services 00 0000 eee A 5 Interprocess dMeSSaP TI paar nahi e eat to m CU Te DI auae A 5 Instrument Settings Changes uuuseeeuuue A 5 Password i Protection 5er xe px dex Eque d wea uices A 5 Minimum Kinetic Interval oon p20 wmvdn mer odd A 5 Autosave C harigess cae bs biben tette Ee aoe oweecewe A 5 AutopritiE C han esra iet broek che ced sapo Ed s Pot a A 5 SoftMax Pro 3 1 1 Update Specification 0 0 0 0005 A 6 Correction of Problems in SoftMax Pro 3 1 A 6 New Interprocess Messaging Features A 6 SoftMax Pro 3 1 2 Update Specification 0 0 0 0 0006 A 7 New and Revised Interprocess Messaging Features A 7 SoftMax Pro 3 2 Update Specification 000000000 A 7 ElexStation Features loras beat eh os tate athe i te ee aimee dee A 7 NEw ACCESSOS 2c dash Au bor eod b DPA E aus soos deb a he es A 8 heck UH soc uee roi NI D Squid e dde A 8 Import EPO earra aa bean wets Gusabidis e dada A 8 PICO BCA id sta staat es ine tute Care Gees Gals A 8 Reduction Modifications ait ed 6505 cer Xa cee Se eee es A 8 SoftMax Pro 4 0 4 01 Update Specification 00 A
73. Box Windows Highlight the folder within which you want files to be saved The Select button at the bottom of the dialog box will reflect this choice clicking the Select button will set this folder as the default location for saved data files You can change the location at any time by repeating this process To change the file format from SoftMax Pro to ASCII text select the Text File format from the list 2 20 SoftMax Pro User s Manual Chapter 2 Installation If the Autosave function is reporting an error or if files are saved in the wrong location it may be that a previous version of SoftMax Pro was not uninstalled prior to installing a newer version Follow the instructions given above to choose the correct folder for saved files Create New File when saving as Text file only After each Plate section or CuvetteSet section is read the data will be saved in a new file This file will contain all the data from the current and previous read ines for example if you read a Plate section then another Plate section fol lowed by a CuvetteSet section Filel will contain data for Plate1 File2 will contain data for Platel and Plate2 and File3 will contain data for both plates and the cuvette This option is available for both file formats SoftMax Pro and ASCII text N KANON Ifyou Autosave to ASCII text format you will not have a SoftMax Pro version of the file unless you also manually save the file using the Sa
74. Boxes for Endpoint Kinetic and Spectrum Modes CuvetteSet Section cece ee 7 26 Example Reduction Dialog Box Showing PathCheck Pre Read and Pae Dank O PUONSs ceceacnet saenoreaes oe ease sesso Stare a 7 27 Different Reductions of the Same Data Using Different MaxOD Points 7 29 Ditierent Lap Lime Serine PEPPER D m 7 30 Kinetic Reduction with Absolute Values Enabled 00005 7 31 Limits in Reduction Dialog Box 0 0 0 eee cee een 7 32 Kinetic Reduction with Absolute Values Enabled and MaxOD Value Increased7 32 Custom Selected Showing Formula Button Endpoint sess 7 38 Wavelength Calculation Dialog Box Endpoint 0 0 e eee eee ee 7 38 Cursor Positioned on a Line between Columns 0000 00 e eee 7 42 Dragging a Column Divider Line ssis deu dirait 9 rpg V US qaem dose ore ies 7 42 The Show Formulas Command in the Group Menu 00 7 43 Formulas Shown in Group Columns 00 cece cece eee eens 7 44 SoftMax Pro User s Manual Figure 7 32 Figure 7 33 Figure 7 34 Figure 7 35 Figure 7 36 Figure 7 37 Figure 7 38 Figure 7 39 Figure 7 40 Figure 7 41 Figure 7 42 Figure 7 43 Figure 7 44 Figure 7 45 Figure 7 46 Figure 7 47 Figure 7 48 Figure 7 49 Figure 7 50 Figure 7 51 Figure 7 52 Figure 7 53 Figure 7 54 Figure 7 55 Figure 8 1 Figure 8 2 Figure 8 3 Figure 8 4 Figure 8 5 F
75. C and Visual Basic modules Excel macros etc An example of retrieving clipboard data is shown below where the string data retrieved is finally stored into a Microsoft CString object SoftMax Pro User s Manual Chapter 12 SoftMax Pro Remote Command Language if OpenClipboard NULL return FALSE Error opening clipboard HANDLE handle GetClipboardData CF_TEXT if handle NULL CloseClipboard return FALSE Error no data in clipboard get clipboard data store in MFC CString object CString returnStr CString char handle close the clipboard CloseClipboard 2 The WM_COPYDATA approach e Call the Win32 RegisterWindowMessage function to register a unique mes sage tag SoftMax Pro uses this tag information when the command is received to distinguish between a Windows generated message and an external command The value returned should be a non zero value UINT tag RegisterWindowMessage SOFTMaxProMsge Call the Win32 RegisterWindowMessage function to register another unique message tag Your software will use this tag information when the asyn chronous windows message is returned and will distinguish between a Windows generated message and an external command The value returned should be a non zero value UINT replyTag RegisterWindowMessage SOFTMaxProReplyMsg All messages must be sent to the SoftMax Pro main window Note if Soft Max Pro is not curre
76. Column Formula Dialog Box In the Column Formula dialog box you can change the name of the new col umn from the default which simply lists the column by number to reflect the data that it will contain If no existing column is selected when you click the Create Column button the new column will be created to the right of all existing ones If you select highlight a column and then add a new column it will be positioned to the right of the one you selected If the table is currently as wide as the sheet of paper specified in Page Setup when a new column is added the columns will disappear off the right side of the window but are still there To bring these columns back into view resize visible columns smaller New columns can contain references to other columns both in the current section or in a different one within the same experiment For example if you wanted to subtract the mean values in one group from those in another you could create a new column in either group to do this If the two groups were named Group 1 and Group 2 for example and both groups contained a column entitled Mean you could create a column within Group 1 that would subtract the mean values in Group 2 from those in Group 1 The column formula needed to do this would be Mean MeanQ Group2Z Numerous possibilities exist for creating and editing column formulas to dis play the data you desire For a complete discussion of formulas and opera
77. Combination Time at Minimum Time at Maximum Time at 172 llaximum Slope Area Under Curve o jJ Lag Time p o i a Absorbance End Time i SETransmittance e Use plate blank _ Absolute Values Cj Custom Reduction S Spectrum P ILm1 Max imum Minimum v Lambda at Maximum ee Spectrum Pree ipy eeseeeereeeresrensrsnsrnssrussrnsrnnnrnnnrunnnunnnnnnnnnnnnnnunnnnnnnnnnAnnNANENANEANNNANNNANNANNENANEANNAANNNANEANNENNNEANNANNNNANEANNEANNEANNANANAANNANANANNEANNEN NENNEN NENE t Lambda at Minimum 2 Lambda at Mas imum wr Area Under Curve Absorbance 2 Co S Transmittance De Use plate blank Figure 7 18 Reduction Dialog Boxes for Endpoint Kinetic and Spectrum Modes Plate Section SoftMax Pro User s Manual 7 25 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Reduction Dialog Boxes Default Reductions Reduction Endpoint Custom v Lm IPathlength a Absorbance C Transmittance Custom Reduction lt lt ILmi Kinetic i y Ymax Ymax Cunits per sec Time to Ymax Onset Time E Time at Minimum Time at Maximum LagTime bo amp Absorbance Time at 172 Maximum End Time 3 Transmittance z Slope v Absolute Values Area Under Curve Spectrum Reduction Wavelength Combination 1 Custom Y Imi Spectrum Peet ara sense ae eee eee ce el dl ees he eels td eed A le Maximum i I1ininium w Lambda at Maximum i OG Absorbance Lambda at Minimum j 2
78. Computer and then press the CD eject button on the CD ROM drive Replace the CD in its holder and store it in a safe place An open folder named SoftMax Pro 4 3 will be visible on your screen This folder contains the SoftMax Pro program If you want SoftMax Pro to be able to communicate with your MDC Microplate System instrument be sure to turn the instrument on prior to starting the SoftMax Pro program After the instrument is powered on and has completed its start up sequence double click the Spf icon to start SoftMax Pro In Windows a shortcut for the program will be available in the Start menu under Programs SoftMax Pro 4 3 Spf An uninstall program is available for Windows computers Should you need to remove older versions of SoftMax Pro from your hard drive using the uninstall program is recommended since it also removes information regarding SoftMax Pro from the Windows Reg istry To remove SoftMax Pro from a Macintosh computer simply drag the SoftMax Pro folder and any other SoftMax Pro files you may have on your hard drive to the trash When you uninstall the pro gram make sure to backup your data to a folder outside of the Soft Max Pro folder because the SoftMax Pro folder will be deleted What Is Installed SoftMax Pro User s Manual SoftMax Pro can create and use two types of programs protocol files and data files Protocol files function similarly to template files opening a protocol fi
79. Delay ln secs Figure 5 3 Instrument Settings Dialog Boxes for Spectrum Mode 5 8 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Well Scan Mode Wavelengths f Number of Wavelenaths E Excitation Emission IV Auto Cutoff yet fas Tv E T Fluorescence RFUs C Luminescence RLUs C Time Resolved RFUs Sensitivity Fast Normal Precise C D ma Instrument Settings L E x amp r7 PMT Sensitivity e od Read Type al Reade ee ird Automix Fluorescence EE MOUNTAIN had Wee teed Aol M LM aU EN IM ral ET LAM Need teed IM EMI anette teed LIS DTE I ree teed D IDE Fluorescence RFUs Before First Read E sees C Luminescence F Wavelengths Ex Em Auto Cutoff Gn 485 538 530 Sensitivity Readings 5 Time Resolved RFUS AUS AutoCalibrate Ranges LEES oo 0E Before Off AutoCalibrate On Well Scan Editar Pattern Fill Density 3 Assay Plate Type 96 Well Standard Cancel Wells To Read Read entire plate AutoRead Off v AutoCalibrate Well Scan Editor Point Spacing 1 13 mm Points Per Well 9 Approximate Read Time 7 minutes Assay Plate Type Wells To Read Select an assay plate type 96 well plates 96 Yell Standard 384 well plates 384 Vell Standard 48 well plates 48 Well Costar 24 well plates 24 Well Costar 12 well plates 12 Well Costar 6 well plate
80. Endpoint Kinetic Automix amp Blanking E E 3 Automix amp Blanking pe Autor i i amp utomix Before First Read j Before First Read _ Between Reads Figure 4 13 Automix Settings Box SpectraMax and VersaMax Instruments Before First Read This setting can be used with any read mode Click the checkbox next to Before First Read to shake the microplate before the first reading Whether you read at a single wavelength or at multiple wavelengths enabling Auto mix for Before First Read will shake the plate for the set amount of time before the first wavelength reading only You can accept the default value shown for the amount of time to shake the plate or enter a different value up to 999 seconds in the box to the right Between Reads SoftMax Pro User s Manual This option is applicable only to Kinetic readings and will be displayed only when Kinetic mode is selected To enable automatic mixing of the microplate between readings during a Kinetic assay click the checkbox next to this set ting For readings at a single wavelength Between Reads will shake the plate for the set amount of time prior to each reading at that wavelength For readings at multiple wavelengths enabling Between Reads will shake the plate for the set amount of time before each reading at the first wavelength only You can accept the default value shown for the amount of time to shake the plate or enter a different val
81. Experiment menu the Statements menu appears it replaces the other section menus such as Plate Notes etc The Statements menu allows the active user to create or modify statements in the current experiment Add Statement Button equivalent Allows the current user to create a new statement if that privilege is available to the current user Available only to users with permission to create modify e statements Can only be performed on unsigned Statements Modify Statement Button equivalent double click the statement Opens the Electronic Statement Setup dialog box allowing the current user to change the title and text of the active statement Available only to users with permission to create modify e statements Can only be performed on unsigned Statements Remove All Signatures If a statement has been signed a user with this privilege can remove all signa tures from the active statement When all signatures have been removed the file becomes editable once more Copy Statement Copies the title and body of the statement onto the clipboard Does not copy information added to a signed statement Paste Statement Inserts the clipboard contents into a Statement section of the current experi ment Available only to users with permission to create modify e statements Can only be performed on unsigned Statements Sign Statement Button equivalent ef Displays the Sign St
82. Fit 7 56 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Log Log The log log function fits the best straight line to the set of data which consists of the logarithm of the readings on the Y axis the response and the logarithm of the dose on the X axis The resulting display will be a straight line with both axes drawn in logarithmic scale The equation for the log log fit is log4o Y A t B log49 X and is shown on the screen as log y A B log x where A is the log Y intercept of the line for log49 X 0 and B is the slope note log in this equation is the common or base 10 logarithm E StandardCurve Fit LogzLog StandardCurve 1 2 a p m Cc wm 2 Concentration LoglyJ amp B Logix o STD Standard Concentration vs PTeanValue Figure 7 45 Graph with Log Log Fit SoftMax Pro User s Manual 7 57 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments l B Quadratic The quadratic function fits the best parabola to the data The parabola is a curved line based on the equation y A4 B x C x which is shown on the screen as ysASXBx4C2 where A is the intercept B is the slope of the curve at the intercept and C is the measure of the curvature of the parabola The quadratic fit is most appropriate when the standard curve has a tendency to curve up or down An example of a quadratic
83. Folder which allows you to choose the folder within which SoftMax Pro will look to find the Default Protocol file when creating a new experi ment At the bottom of this menu beneath the dividing line is a list of all pro tocol files contained in the currently chosen folder Assays Set Folder protocol 1 Figure 9 1 The Assays Menu To have SoftMax Pro look in a different folder for the Default Protocol file choose Set Folder A dialog box will open allowing you to locate and set a different default folder Figure Figure 9 2 shows the dialog box that appears for the Macintosh and Figure Figure 9 3 shows the dialog box that appears for the Windows environment L3 SOFImaxie PRO x Avalon Select Assays Figure 9 2 Set Folder Dialog Box Macintosh 9 3 Chapter 9 File Management and Printing 9 4 Select the folder you wish to use and then click the large button at the bottom of the dialog box to set this folder as the default The name shown in this but ton will change to reflect the name of the selected folder NOH Molecular Devices recommends that you have only one default protocol Select Directory Directory c szaftmas pro Co Assays zb Ses Drives NN E c C Figure 9 3 Select Folder Dialog Box Windows it Locate and highlight the folder you wish to use and then click the Select cr gi button at the right of the dialog box to set t
84. Graph with Points Connected Linear Fit Applied Bar Graphs Cluster and Stack 0 cece eens X Axis Dialog Box Relationship of Sections Plate Section Endpoint Kinetic Spectrum Well Scan and FlexStation Display Dialog DOX6SL 4istucsiends adag PLE d 1 RUP Re e UR eek depen es Choices for a 384 Well Plate Display Normal Vertical Rotated Large and Interleaved Default Displays for a Plate Section Display Dialog Box for Kinetic Threshold Display Kinetic Data Displayed as Threshold with Reduced Number Kinetic Data Reduced Displayed as Ranged ssleeeseeeeee ees Endpoint Data Reduced Displayed as Gray Scale 00000 e eee Graph Options DID ocio iur eraut doomed eee arduus Gr E Rapid ddr Well Graph for a Single Kinetic Microplate Well Raw Data Well Graph for Three Kinetic Wells of a Microplate Raw Data Masked Wells in a Plate Section Reduction Dialog Boxes for All Modes 0 0 cece eee eee Different Reductions of the Same Data Using Different MaxRFU Points Ditterent Lae Tine SetLlfig S osea a mracitas qoc NEU RE Iop o aed prices E Kinetic Reduction with Absolute Values Enabled Limits in Reduction Dialog Box RFU shown Kinetic Reduction with Absolute Values Enabled and MaxRFU Value Increased FLEX Mode Reduction Dialog Box Smoothing Setting FLEX Display Options showing Baseline option 00 ee eee FLEX Mode Reduction Dialog Box
85. Headed bfe anale Activa Arrow STACTS Pointer eo Dividing E 1008 04 Line HOS A eiu Dataos 1817 40 1017 4 i 1939 554 2111 08 Dragging dus 2067 10 AG 1989 73 Right 1889 72 1528 52 1828 52 e24 450 874 073 265 303 42 8 Resulting 677 540 1005 87 Column 1S ese ars Wider 807 785 877 000 853 381 SSSA 1008 04 o Data 14917 40 1938 854 58575 83 2111 08 2067 10 Figure 10 70 Sizing the Data Group s Concentration Column Wider To shrink this column to the minimum size needed to display the complete title click once in the Concentration column to highlight it Then choose Autosize from the Group menu The column will be reduced in width as shown in Figure 10 32 Several columns can be Autosized at one time by highlighting dragging across multiple columns in the Group and then choosing Autosize SoftMax Pro User s Manual 10 59 Chapter 10 Tutorials 874 073 26 303 D ataj f 1939 554 58 575 g 3 73 Before jon Concentration Concentratio 874073 26 303 Datad3 0 050 1939 854 58 575 Figure 10 71 Columns Before and After Autosize C Add a Column The columns that are created by default with various Group sections may not always meet all the needs of a particular experiment You can add a column to any Group section and define the purpose of this column in your data analysis By default new columns are added to the right of any selected hi
86. High Medi S S b UU uu blank time field 9 20 SoftMax Pro User s Manual 1 3 Chapter 9 File Management and Printing Table 9 2 ASCII Data File Fields and Descriptors End integration time Time to end integration for time resolved fluorescence otherwise blank field 29 First row Firstrowread First row read 1 to max First row read 1 to max alwayslforcuvettes 0 for cuvettes Last row read Eu C to mao always I forwettes to max always 8 for cuvettes Time tags flag Indicate whether the exported data has read time tags The information for the first plate or cuvette section is provided in the lines fol lowing the descriptors The number of lines of data depend on the export format and the type of test that was run A minimum of one line of data is exported when the Time format is used with a single wavelength Endpoint read If the instrument you are using has a temperature associated with the data all MDC Microplate System instruments except Emax Vmax and UVmax the temperature at the time of the reading will appear before the first data point in the reading The second line of a file for a Group table section includes fields that corre spond to the number and types of columns found in that section they are labeled as the table is labeled and look very much like the table itself Following the data a blank line is included and then a line with a single ter mination field End Each subse
87. In this display the wells are shown in a format that skips every other well as follows all odd columns and rows begin in the upper left corner of the plate display and are followed by all even columns and rows So well A1 is still in the upper left corner but it is followed horizon tally by well A3 A5 A23 A2 A4 etc and vertically by well C1 E1 O1 B1 D1 etc This display is most useful when the 384 well plate is composed of 4 daughter plates of 96 wells each Figure 8 4 shows the default displays for Endpoint Kinetic and Spectrum modes The default display for Endpoint mode is the raw fluorescence lumi nescence For Kinetic Spectrum and FLEX modes the default display is raw RFU RLU shown in plot format RFU RLU vs time for Kinetic and fluidics and RFU RLU vs wavelength for Spectrum scans Well scan data defaults to a display as shades of gray Reduced Number The reduced number is based on the settings defined in the Reduction dialog box It is a combination of plate blank subtraction in the Plate section wave length reduction and either a Spectrum or Well scan or a Kinetic or FLEX reduction The information acquired from the instrument is reduced to a sin gle number or value The reduced number will be reported in the Group sec tion when a template has been defined To view by reduced number alone select the Number option the first choice to the right of the default just after the dividing line to
88. Management and Printing File Protection Individual protocol and data files can be protected from changes Two types of protection are available e Password Protection avoids changes being made to a protocol or data file by persons who do not know the password and provides an electronic sig nature with the file e Shielding a File preserves the integrity of data in a file by prohibiting any changes to the file with the exception of adding or changing text or sum maries in a Notes section Additionally shielding requires both an author s and user s name Password Protection When a protocol file is password protected you may read data into the file but may not change it in any other way without entering a password When a data file is password protected you may view the file but are unable to make changes without entering a password Password protecting a file requires that you enter an author s name and password Table 9 5 shows the actions that are protected when a password is assigned to a SoftMax Pro file SoftMax Pro User s Manual 9 29 Chapter 9 File Management and Printing Table 9 5 Actions Protected by Password Menus ee Dialog Boxes Others Buttons File gt Page Setup Moving a formula in a Notes section File gt Import Resizing columns in a Group File gt Save Template Editor Moving sections and experiments Reduction Typing in sections Notes Plate and Cuvette Set Display Mask Well Ne
89. Once linked to SoftMax Pro Enterprise Edition by the administrator the database in Enter prise Administrator is referenced to validate the user name entered in Soft Max Pro and password entered and to retrieve privilege information for that user The combined capability of these software programs provides a full audit trail that lists all user activities User privileges set within Enterprise Administra tor determine the extent of user activity which menu choices are available etc for that user within SoftMax Pro Enterprise Edition A user who has not been added to the database may log in to the Enterprise Edition as a guest Guests may open and view existing files but may not modify them Within the Enterprise Edition a privileged user may add an Electronic State ments section to an experiment Electronic statements provide a means of des ignating that an experiment has been approved reviewed or verified Privileged users may also apply electronic signatures to these statements Once a statement has been signed the file may not be modified Electronic statements and signatures are saved with data files but signatures are NOT saved with protocols Only one Statement with signatures is allowed per file The Statement section is always located as the first item of the Experi ment before any Notes section Files created in the Enterprise Edition of SoftMax Pro will show the filename extensions of epr for protocol files or ed
90. Opens the Instrument Settings dialog box for this menu plate Setup Opens the Template dialog box allowing you to create or edit the template EJ Template Opens the Reduction dialog box containing reduction settings Reduction Template in the Plate menu Reduction in the Plate menu Double click in a single well or Enlarges selected well s to a Well Graph display when several wells are selected Kinetic and Spectrum modes only choosing Graph in the Plate menu Graph again returns to regular display toggle Opens the Display dialog box containing display Ej Display E Display Display in the Plate menu E R Mask in the Plate menu Masks the selected wells Printer icon Includes excludes section from printed report Opens the Section dialog box allowing you to Section Name in the Plate menu rename the section New Window in the Plate menu Opens the section in a new window Copies the template allowing it to be pasted to Copy Template in the Plate menu B E den Pastes a previously copied template to a new Paste Template in the Plate menu Piate decion Export Template in the Plate Opens the Export Template to dialog allowing menu you to export a template to an ASCII text file Import Template in the Plate Opens the Import Template to dialog allowing menu you to import a template from an ASCII text file Uo 16 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview
91. Plate section only Wells can be assigned to a Blank group in the Template Editor The average value of all the wells in a plate blank group is subtracted from all individual well val ues obtained during the reading of microplates Plate Blank is applied to raw data Plate Blank subtraction may be turned off in the Reduction dialog box so that the data may be seen with or without plate blank subtraction Plate blanks are only subtracted from wells on the same plate to which they are assigned The checkbox for Plate Blank subtraction only appears in the Reduction dialog box after data has been col lected For Kinetic and Spectrum data the blank value is averaged and subtracted on a point by point basis The example below shows a graphic representation of original and average plate blank data followed by the resulting data with average subtracted RLU RLU RLU Time Time one Blank Subtracted Sample Well Average of Blank Wells Sample Well Figure 5 30 Graphic Representation of Plate Blank Subtraction Group Associated Blanks Blank wells can be assigned within any group other than the Blank group in the Template Editor The average reduced value of the blank wells within that group will be subtracted from individual reduced values within that group only This blanking function uses the reduced number example Lm1 Lm2 Vmax etc only it does not subtract on a point by point basis When a group blank has been assig
92. Pro User s Manual 5 13 Chapter 5 Reading Using Fluorescence or Luminescence Instruments measure fluorescence after the lamp is turned off Time resolved fluorescence is used to reduce the amount of background noise which interferes with fluo rescence The excitation lamp flashes and after it is off the delayed emission is collected for a set period of time before the lamp is flashed again Bottom Read FlexStation Instruments Only When a FlexStation instrument is connected to the computer or chosen in the Preferences below the Read Type choices is a checkbox next to Bottom Read When selected Bottom Read allows the FlexStation instrument to read through the bottom of clear bottom microplates By default this setting is enabled for Well Scan and FLEX modes and off for the others This setting is dimmed if a plate type having an opaque bottom is chosen Wavelengths For Endpoint Kinetic or Well Scan Readings Choose the number of different wavelengths to be read At least one wave length must always be specified Selecting more than one wavelength will cause a corresponding number of wavelength setting boxes to appear maxi mum four wavelengths Wavelengths Hibernia teense Excitation Emission Auto Cuttaff um fos I Be qp 55 wz fe dm fo 59 Figure 5 9 Wavelength Settings The wavelengths specified previously or the defaults will be shown in the boxes You can choose a wavelength from the
93. Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments 5 1 O ono Mr rm 5 3 InsStrment Selng8 s iet uper Sup dnt tace ipae pda bra te gd 5 4 Instrument Settings Options 0 0 0 cee eee eee 5 10 Template POUOl TE 5 19 Selecting Wells in the Template Editor 5 21 The Template Editor Tool Bar 0 0 0 eee eee 5 21 ui RP H 5 30 Copying and Pasting Templates 005 5 33 Rgadino a Mviruro oc Rr 5 34 General Information about a Reading issuu 5 35 Reading Microplates Using the FlexStation Instrument in PELA MO P r H 5 36 Instr nie HE SCLIN GS oa etes aa dor ponerent Y ip eee e mardi erates 5 38 Rea Ode 3 Spe een pn dy a soe oman Vana ene eens 5 40 Deis LOr FLEA MOUSE aeger ata empto eco antedicte reb a 5 41 Template ECO 2a corr deo 9 iaiT adni SUR reed 5 51 IRCA Cn a Microplate caresser derit Ea doo dud aUe eredi 5 51 General Information about a Reading 0 5 52 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator i42 sea ds cae dd 444r a ad rr ER R 6 1 PAED DETTO 6 2 General Information 20 lt 0 lt s et erue rrt rh ren 6 2 Enterprise Administrator au dario uo d Ven Eo Hd RURAL RADAR qon yao Qd 6 3 Preinstallation Checklist llssleeeleeeeessse 6 3 Pre Installation Information 0 0 00 e ee eee 6 5 General
94. Pro and it s utilities Software included with SoftMax Pro upon license The user is licensed to copy the software for a backup or archival copy as necessary Simultaneous use of the software with more than one MDC Microplate Reader System per license is expressly prohibited Simul taneous use of the software on four 4 computers solely for the purpose of analyzing already acquired data is expressly permitted The user is expressly prohibited from using the software to control any other instru ment computing hardware or peripherals other than MDC Microplate Systems This license agreement does not grant the user title to the software or any copyrights of proprietary rights in the software The user may not sublicense rent lease modify translate decompile or disassemble the software for any purpose The User however does own the media on which the software is originally or subsequently recorder or fixed MDC retains title and ownership of the software recorded on the original disc and all subsequent copies of the software regardless of the form or media in or on which the original and other copies may exist This license is not a sale of the original software or any copy LIMITED WARRANTY MDC warrants that the disc on which the program is furnished is free of defects in materials and workman ship and will operate under normal use for a period of 90 days from the date of delivery Except as provided above the Software is provided as i
95. Protocol Tutorial This protocol contains this introduction as well as one Experiment section If you are new to SOF Trax PRO follow thus tutorial as outlined m Chapter 7 of the User IvTanual Experiment 1 Quantitative Endpoint assay with Standards and Unknowns with and without dilution factor The unknowns are Interpolated from a standard curve To customize the Default Protocol First delete any sections you do not want for example this introduction second make any changes you want to the mstrument setup Last save the default protocol with the name Default Protocol in the same folder as the SOFT max PRO application you will be asked if you wish to replace the existing default protocol suy cH Experiment 1 b Es Plates Standards Hot 24 gt Controls m B gt Samples ua E fta Samw di HeH E4 b Z Granit Ft p ff cuvetesew mj e m e e e Je fn Figure 2 6 SoftMax Pro Default Protocol Untitled Window SoftMax Pro User s Manual 3 Chapter 2 Installation Communicating with the MDC Microplate System Instrument After installation of SoftMax Pro you will note that the instrument icon in the upper left hand corner of the window appears with an X and the words No port selected through it You must access the Preferences settings from the Edit menu choose the Preferences command to select the proper com munications port which port depends on the configuration of your com puter
96. ReadsMVell 6 Lag Time 0 00 End Time 5 00 RFU Min 0 RFU Max 20000 Ymax Pts 34 34 Plate Last Read 2 55 PM 1 22 2001 Figure 8 4 Default Displays for a Plate Section SoftMax Pro User s Manual mma E Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Threshold Ranged and Gray Scale SoftMax Pro User s Manual To display Threshold Ranged or Gray Scale data high and low values must be entered When you choose one of these display options SoftMax Pro pro vides default values for the high and low limits by means of an autoranging function encompassing all the data from lowest to highest value You can change the high and low limits by highlighting the existing value and enter ing a new value An example of the Display dialog box for a Kinetic reading Plate section with the Threshold display option chosen is shown in Figure 8 9 wm Display ES Raw Reduced Ymax units per sec Lm Example Options cee ee ene DM EN Display Threshold wr ou High Low Limit Boxes Show wwith reduced number Cancel Figure 8 5 Display Dialog Box for Kinetic Threshold Display Threshold The Threshold display shows the raw data as a plus for values above the high limit an asterisk for values within the limits and a minus for val ues below the low limit Figure 8 6 shows an Endpoint reading displayed using the Threshold option along wit
97. Spectrum scan or Kinetic reduction The informa tion acquired from the microplate reader is reduced to a single number or value The reduced number will be reported in the Group section when a tem plate has been defined To view by reduced number alone select the Number option the first choice to the right of the default just after the dividing line to see a reduced num ber as well as another type of display click the box next to the option that says With reduced number in the lower left of the dialog box to enable it Chang ing the reduction parameters after choosing a reduced data display mode will cause the display to update accordingly More information about reduction formulas and how to customize them follows later in this chapter SoftMax Pro User s Manual L d Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Threshold Ranged and Gray Scale SoftMax Pro User s Manual To display Threshold Ranged or Gray Scale data high and low values must be entered When you choose one of these display options SoftMax Pro pro vides default values for the high and low limits by means of an autoranging function encompassing all the data from lowest to highest value You can change the high and low limits by highlighting the existing value and enter ing a new value An example of the Display dialog box for a Kinetic reading Plate section with the Threshold display option chosen is shown in Figure 7 9
98. Transmittance rea Under Curve Custom Figure 7 19 Reduction Dialog Boxes for Endpoint Kinetic and Spectrum Modes CuvetteSet Section The PathCheck for those instruments that support this function and pre read plate reduction parameter checkboxes will not be displayed in the Plate section Reduction dialog box unless they have been selected in the Instru ment Settings dialog box Figure 7 20 shows how the Reduction dialog box looks with the Use plate blank Use pre read plate Switch normal and pre read and Apply PathCheck options available 7 26 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Endpoint Reduction Ej Use plate blank i Absorbance De Use pre read plate T Transmittance Switch normal and pre read i wf Apply PathCheck Figure 7 20 Example Reduction Dialog Box Showing PathCheck Pre Read and Plate Blank Options All of the read modes offer one or more predefined choices for reduction The predefined reductions are described below under the appropriate read mode heading In addition with any read mode you can create a custom reduction formula to better suit your needs Since all custom reduction formulas are defined the same way regardless of read mode they are described together after the individual read mode sections Wavelength Combination The default reductions for Endpoint readings are
99. Update Specification 000 A 9 GLP GMI SHDDOERE eus PERO Pra sade a A A 9 Pte OM CS oT A 9 SoftMax Pro User s Manual SoftMax Pro User s Manual PILAR MICS oe ose geans PT EPUPUE V EIUS dp EA uem ORE d oy oe A 9 ISODOHOS aseua stor uo tora d gehe qe ROSEO edd fedus Ibid fert A 9 FlexStation Enhancements 0 eee ee eeee lees A 9 Vell cand Mode ved ud uhi eire bus aes S aet Rub anii Modi A 10 Export Function Updates vvoed eroi rp dh A 10 Multithreading of Serial Input PC only A 10 Macintosh Communications Toolbox Support A 10 SpectraMax Gemini and Gemini XS Enhancements A 10 Enhanced 4 Parameter Fit lslsls esses A 10 Documentation amp Software Enhancements A 10 eelan aere a AE andae ad Vl a NU IF re ere A 10 SoftMax Pro 4 15pecification lesse A 11 DDECITICHIOTIS o aur mcg eun ina give a dpsag Ges a ua Hr s apo Ra A 11 Appendix B Glossary of Terms cece eee eee eee B 1 TETTE I 1 xi xii SoftMax Pro User s Manual Figures Figure 1 1 Figure 1 2 Figure 1 3 Figure 1 4 Figure 1 5 Figure 1 6 Figure 1 7 Figure 2 1 Figure 2 2 Figure 2 3 Figure 2 4 Figure 2 5 Figure 2 6 Figure 2 7 Figure 2 8 Figure 2 9 Figure 2 10 Figure 2 11 Figure 2 12 Figure 2 13 Figure 2 14 Figure 2 15 Figure 3 1 Figure 3 2 Figure 3 3 Figure 3 4 Figure 3 5 Figure 3 6 F
100. all wavelengths and visibly inspect the plate data for missing readings Lost Data with a Single Wavelength Kinetic Reading If the error occurs during a single wavelength Kinetic run access the Reduc tion dialog box click the Reduction button for the plate section that was read Choose Custom for the Wavelength reduction and then click the for mula button that appears f to bring up the Reduction dialog box For the Kinetic reduction enter the formula numpointsread and then close the dia log box In the Plate section set the display click the Di spl ay button to raw data plot with reduced number Compare the number shown in the Plate display with the number of Vmax Points listed at the right hand side of the Plate section The numbers should be the same If a time point was not col lected in a well the Plate display number will be lower Lost Data with a Multiple Wavelength Kinetic Reading If the problem occurs during a multiple wavelength Kinetic run you can fol low the procedure outlined above changing the wavelength reduction in turn to each of the wavelengths read Lm1 Lm2 etc and then checking the num ber of points read at each wavelength Once you know which wells have a missing time point you can look at a Well Graph with points connected and determine if the missing points will adversely affect data analysis SoftMax Pro User s Manual Chapter 11 Troubleshooting One or more points may have be
101. an Endpoint Reduction dialog box is shown in Figure 8 23 Custom reduction formulas may contain other operators and terms in addition to wavelengths Wi Reduction E X nene Wavelength Combinatiorr egal Weed meten meme cea meten ane eC Dee Pe SR POS AT Phe POR gue ved yee eget eed Dh e REDDERE ETATE ger E Er C EE IE EI ILE cc E E E DE EE Formula Exec onto feni factor eei feci fornisce forsan ge c oec Roe eec eec ar ayn piston ah ioc ey Ecce i nic iar akong righ Hore ec eee erc eis tee piston cio aa torigetae gun eei egi esce rci ecc barr eei Kec Rec eec fora ic ec a cres c Rc Er Reg ae DRIFT RS ET EE Button Mnm r fo Latime bo r Absolute Values Max RFU 20000 End Time E pen eene enn enne eng en vien pec enn onnpean eng enn peer ean enne penne ean een Figure 8 23 Custom Selected Showing Formula Button Endpoint Clicking the Formula button opens the Calculation dialog box in which you can name and define a new reduction formula Calculation a Accent Figure 8 24 Wavelength Calculation Dialog Box Endpoint Examples of some formulas that might be used when combining multiple wavelengths in the Calculation dialog box are given in Table 8 3 Table 8 3 Wavelength Reduction Formula Examples Lim1 Lm2 Lm1 Lm2 Lm3 Lm1 Lm2 Lmn Lm1 Lim2 ILim1 Lm3 ILim2 ILim1 Lm6 ILim2 Lm5 Lm3 Lm3 Lm4 Lm1 Lm2 Lm1 Lim3 Lm1 Lmn eae Lm2
102. and Analyzing Data Using Fluorescence or Luminescence Instruments 1 k 6 28 Onset Time This is another method for analyzing non linear Kinetic readings Onset Time reports the time required for a Kinetic reaction to change by a specified amount onset RFU RLU above the initial RFU RLU reading for the well This elapsed time data is useful for cascade reactions including clot formation endotoxin testing for example or clot lysis applications where the change in reagent concentration does not affect the maximum optical density change but changes the time required for the reaction to reach completion Time at Minimum This setting reports the time at the minimum RFU or RLU that falls within the reduction limits Time at Maximum This setting reports the time at the maximum RFU or RLU that falls within the reduction limits Time at 1 2 Maximum This setting reports the time at the half of the maximum RFU or RLU that falls within the reduction limits To calculate this reduction SoftMax Pro first determines the point within the reduction limits that has the maximum sig nal level RFU or RLU It then scans the plot from left to right until it finds two points having signals that bracket half of that value A linear interpola tion between these two points is used to estimate the Time at 1 2 Maximum Slope This setting determines the slope of the combined plot i e the slope of the line using linear regression after the wa
103. and modify e statements C Clear Audit Trail a Change Autosave parameters C Change Group Table and Template settings C Change Data Reduction Settings C Change Instrument Settings C Change Notes Text C Mask UnMask Wells Read Empty Plate and Cuvette Sections L Overwrite Plate and Cuvette Data a Remove signatures from e statements lt Back Cancel Help Figure 6 4 User Privileges Dialog Box Choose Group Using groups pre defined settings can be applied quickly and easily to a new user Defining a group is not required privileges may be set individually Save files and protocols Allows a user to save SoftMax Pro EE files to disk Sign e statements A user can e sign statements within SoftMax Pro EE Create and modify e statements Allows a user to create and modify statements in an experiment SoftMax Pro User s Manual Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator Clear Audit Trail Allows a user to erase the Audit Trail window that displays all activity within the SoftMax Pro EE program since the program started Change Autosave parameters Allows a user to modify the Autosave features of SoftMax Pro EE Change Group Table and Template settings Gives a user the ability to access and edit the features of the Template option of the Plate menu Change Data Reduction Settings Gives a user the ability to access and edit the features of the Reduction option of t
104. at 515 3 readings PMT set to High Time 200 seconds Interval 2 seconds Total number of readings 101 Minimum interval 1 28 seconds Off Once 96 Well Standard clrbtm A1 H10 Beckman 96 2 3 mL None initial volume 0 uL Not Used Full Rack Compound amp Tip Columns None 10 36 SoftMax Pro User s Manual Chapter 10 Tutorials Define the Protocol The following steps will guide you through the process of creating a protocol file for an EC50 assay using FLEX mode Reading an actual plate will not be required you will run a simulator program to create data and also open a saved data file to compare your file setup and results Step 1 Start SoftMax Pro Create a New File Double click the SoftMax Pro icon to start the program If SoftMax Pro is already running choose New from the File menu You should see a new file labeled Untitled The figure below shows an example of the Default Proto col file w L5 Experiments Qo v E notes fta a Default Protocol Tutorial 2 If you are new to SOFT max PRO follow this tutorial Tutorial 2 as outlined in Chapter 10 of the JOFTmax PROC User s Manual You can also read Chapter 6 Reading Ivhcroplates Using the FLEX station Instrument and Chapter 8 Displaying and Analyzing Data Using Fluorescence Instruments and refer to the Formula Reference Guide to utilize the full potential of SOP Tmax PRO Directions on how to import amp nalyst AcquestiCntenon a
105. at two wavelengths and choose to take a ratio of those wavelengths smoothing would be performed on each individual wavelength and the wavelength ratio would be calculated using smoothed data 8 33 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments l k 8 34 Reduction Baseline Options Three choices that are available within this section of the Reduction dialog box affect the way that the data is graphed e Absolute Shows the raw data without any change in the way it is graphed Raw data is used in subsequent reduction calculations e Zero Baseline This option reduces this interference by offsetting the graph slightly C Abszolute C Zero Baseline Points amp Baseline Mutiplier Se Figure 8 20 FLEX Display Options showing Baseline option If you enter 5 for the number of points for example the average of the first 5 points will be used as the baseline value and all raw data will be sub tracted from this value It is important to note that the bseline subtraction is applied only to the reduced data raw data plots do not change if you wish to view the plot with baseline subtraction you must choose the Reduced Plot button in the Display dialog box or click the Show Reduced button in a zoomed well graph window If two or more wave lengths are present in a reading and you choose to combine these wave lengths while Zero Baseline is selected the zero baselin
106. available clean tip and untargeted compound column rather than reusing tips and compound col umns targeted by the preceding fluid transfer and e Each fluid transfer will use a new tip The algorithm assumes that all of the columns in the Wells to Read area will receive fluid during the initial transfer so the Tips target grid will be filled from left to right starting with the first available tip column and incrementing the Tip target by one until all columns in the Wells to Read selection have been filled In the illustration above the Wells to Read selection includes columns 1 through 4 with an 8 column compound plate and a full rack of tips From those settings SoftMax Pro derives the first transfer s Tips and Compound targets at 1 4 The second transfer targets the first unused Tips and Com pound columns used during the first transfer and the third transfer targets the first unused Tips and Compound columns used during the second trans fer So in this example the second transfer event s targes for both Tips and Compound are 5 8 the third transfer s events are 9 12 5 49 Chapter 5 Reading Using Fluorescence or Luminescence Instruments 2 50 When the number of columns in the Wells to Read area is the same or greater than the number of columns of both Tips and Columns the algorithm fills the Tips and Columns targets as before that is each assay plate column is tar geted with a new tip column and new compound column
107. be controlled by other Windows applications by means of an ASCII string based messaging system Commands are available for opening and saving files controlling instruments selecting sections exporting data from Plate CuvetteSet and Group sections and importing Templates The available commands for interprocess messaging are described in Chapter 12 SoftMax Pro Remote Command Language along with examples for pro gramming these commands 3 40 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Miroda HOT 6 aos Sat eec ve yo eR Ito tease 4 3 Instr rnent DEMING 8 vog exu Sa d beds Sato edad on Sra tus at ero Ed 4 4 Instrument Settings OpUOnS e rRILPVE NEXU e 4 9 lemplate EXE e coke bia Eoo pP pe EL ONCE Am pu Gb ets 4 20 Selecting Wells or Cuvettes in the Template Editor 4 22 The Template Editor Tool Bar 0 2 eee sees 4 23 PANKIN oce opus ey DET ES es Gates iet 4 33 Copying and Pasting Templates 0 0 4 35 Exporting and Importing Templates 4 37 Reading a Microplate or a CUVEE creis erita dints eee eee eee 4 39 Redding a VIcroplate s rast erki Cee ed IS DEB US ENS 4 40 Reading a Cuvette SpectraMax Plus and Plus 9 only 4 4 General Information about a Reading 4 43 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments t 4 2 SoftMax Pro User s Manual
108. button on the tool bar to display the data from multiple wells in one enlarged Well Graph The data in the enlarged Well Graph will update to show new data points as they are received from the MDC Microplate System instrument Other choices and settings within SoftMax Pro affect the way in which data is displayed the type of reduction specific time settings and so on These set tings are discussed in Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 4 45 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments t 4 46 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments IDtt OO te HOT 6 aos Sant eee c e oe eR f tonis eei odes 5 3 Instrument DEMING 8 vog esee Sa d beds n Sato edendo Sri tus ated eed 5 4 Instrument Settings ODIOnDS es 4925 st ieee E Nardi 5 10 lemplate EO e ri trap RERO HR RP pea iere eee 5 19 Selecting Wells in the Template Editor 5 21 The Template Editor Tool Bar 0 2c eee eee ee 5 21 BANKIR aes e e oEyubU oy DOT ES EN ey sees delete 5 30 Copying and Pasting Templates 0 04 5 33 Readies d MictOp late se ses siete Ree ei ewer RE ES 5 34 General Information about a Reading 5 35 Reading Microplates Using the FlexStation Instrument in FUSE IN OA us eosdem E WERT BA OUR o debes 5 36 Instrament Setups cvesa epa Ev tas x EE PES Cue uie pn 5 38 REA MORE 22 15 50
109. cece eee cece eee eee 10 26 Standards Group Section with Formula Shown for Highlighted Column BackCalcConc 10 26 Sizing the Standard Value Column Wider 0 2c eee e ee eee 10 27 Columns Before and After Autosize 00 0 eee cece ee eee 10 28 Calculation Dialog DOX 23 23 92 8457099 G9 aa Pe qdaiq ei EEE cena de dd 10 28 New MaxValue Column Appended to Group Section 10 29 Summaries in the Standards Section 00 c eee eee eee ee eee eeee 10 30 Calculation Dialog Box from Summary in Standards Section 10 30 Standard Section Before and After Hide Replicates 00 10 31 Standard Curve Graph Section No Fit Applied 0000005 10 32 Standard Curve Graph Section with Linear and 4 Parameter Fits Applied 10 32 Graph Options Dialog Box ssseeeeeeeeee e 10 33 Print Dialog Box for Macintosh 0 cece eect eens 10 34 Print Dialog Box for Windows 0 0 ee cee eens 10 34 Default Protocol with Notes Section Open 0 e cece eee eee eee 10 37 Instrument Settings Dialog Box Showing Wavelengths Settings 10 40 Instrument Settings Dialog Box Showing Sensitivity Settings 10 41 Instrument Settings Dialog Box Showing Timing 0000 10 42 Instrument Settings Dialog Box Showing Automix 00 ee eee 10 42 Instrument Settings Dialog Box Showing Wells To Read LLLse 1
110. choices for the three modes raw optical density values numbers for Endpoint e the change in raw OD values over time for Kinetic and e raw OD values for the range of wavelengths displayed as a plot for Spectrum Other choices available in this dialog box include displaying the data as a sin gle reduced value number in threshold ranged or gray scale formats or any combination of display choices with the reduced number the reduced number display choice cannot be used with the reduced number checkbox SoftMax Pro User s Manual d Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Endpoint mw Display EA m Display Ej a n c ie Faw Feduced Raw Faduced Raw OD Reduction Lm1 Example Example Reduced dxsocosaocosconogonasconodonccconcecootenosos Pete ee ee eee eee eee eee eee ree Display Choices Jo M v Humb Raw OD Thresh ld default Ranged Grayscale Vith reduced number Cancel Cancel Kinetic mi Display mi Display E E Exw Reduced Raw Reduced Raw OD Ymax units per sec Limi Example b Raw Data Example Options Exe eres Plot OD v Mumber Display vs Threshol Choices Wavelength Ranged Grayscale EEE EA RUN AEE EE default PETE E E se Show wwith reduced number Cancel Spectrum mi Display Ej Wi Display E x Raw Reduced Raw Reduced Raw OD Lambda at Maxim
111. chosen password Log Out Terminates the user s connection to the SoftMax Pro Enterprise Edition pro gram Change Password Opens a dialog box allowing the current user to change his her password Show Audit Trail Displays a list of all user activities performed on the current file Clear Audit Trail Erases all information shown within the Audit Trail window Show Admin Info Displays the privileges set for the current user 6 16 SoftMax Pro User s Manual Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator User Information Mame Charles Lexington Admin Group C Malecular DevicessSOF TmaxPRO Program File REDatabase edb Permissions t Save files and protocols Create and modify e state ments i Sign e statements Read Empty Plate and Cuyette Sections Change Instrument Settings Change Group Table and Template settings Change Data Reduction Settings i Mase Unhiask Wells Change Hotes Text Change Autazave parameters Figure 6 9 LIser Information Dialog Box E Statements Add Statements A Statement is a section within SoftMax Pro Enterprise Edition only that pro vides a location for statements regarding authorization review and valida tion of data files After a statement is created it can be signed a process that locks the file preventing further changes Add Statements is located under the Experiment menu Selecting Add Statements will open the Electronic State
112. color SVGA monitor e Video adapter and software driver capable of displaying 800 x 600 minimum resolution set to 16 bit high color and small fonts or better e 256 MB RAM or greater e 6 GB hard disk drive e CD ROM drive e Serial port COM1 COM9 Windows Minimum System Configuration e Pentium II 300 MHz processor e Microsoft Windows NT 4 0 or XP e 17 inch color SVGA monitor e Video adapter and software driver capable of displaying 800 x 600 minimum resolution set to 16 bit high color and small fonts or better e 128 MB RAM e 2 GB hard disk drive e CD ROM drive e Serial port COM1 All of these configurations require a straight through serial cable or USB con verter More about the types of cables needed for the different instruments follows in this chapter Molecular Devices recommends using only high qual ity double shielded straight through serial cables If you plan to use SoftMax Pro with a FlexStation instrument or with any instrument if you will be creating large files in excess of 20 plates per file or if you will be using any robotic applications Molecular Devices recommends that you increase the amount of RAM in your computer to a minimum of 256 MB SoftMax Pro User s Manual 2 7 Chapter 2 Installation Install SoftMax Pro 2 6 1 2 3 4 5 6 Three versions of SoftMax Pro are available Life Sciences Drug Discovery and Enterprise Edition The Life Sciences version provi
113. data or mechanical damage It is also used to alert against unsafe practices AA AARNA Used whenever an action or condition might potentially cause serious injury to yourself or to others who might use the instru ment or computer 1 6 SoftMax Pro User s Manual Chapter 1 Welcome to SoftMax Pro Getting Help Macintosh On Line Help SoftMax Pro User s Manual On line help is available while working with SoftMax Pro for the Macintosh by clicking the Help menu at the far right of the menu bar i Click here About Balloon Help for the Help Menu Show Balloons SOFTmax Pro Help SoftMax Pro Help Options Formula Help Figure 1 1 Help Menu for SoftMax Pro for the Macintosh Choosing either of the two help options at the bottom of this menu SoftMax Pro Help or Formula Help causes a Help window to appear This window provides a list of topics on the left and information about the selected topic on the right Figure 1 2 shows the window for Formula Help In this figure the topic Math Functions has been selected on the left and the corresponding information about that topic appears on the right Formula Help Formula Help Topics Math Functions Comparison Operators a NOTE All math functions are calculated in radians 180 m e Conditionals radians Direct Well Accessors Errors Abstnumber Group Accessors Returns the absolute value of a number or list of numbers Interpolation
114. data in a bar graph or another format by clicking the graph options button which opens a dialog box like that shown in the fol lowing figure Graph Options Title Graph 1 Type onts Sw Scatter Sample Text Sel Ob C2 Cluster bar sample Text Set hil Stack bar Legend dnd Connect points O E sample Text Set Del Plot symbols 5 Concentration bats VY 95S ActivationiData Errors Mo Error Y Errors Mo Error Figure 10 78 Graph Options Dialog Box SoftMax Pro User s Manual Chapter 10 Tutorials This dialog box allows you to choose rename the graph choose the type of graph that will be shown and to format the text used in the graph You can add or remove plots click the or Delete buttons and you can also edit existing plots Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using provide more information regarding the graphing options that are available In this example choose the option for Cluster bar and then click OK The resulting graph should look similar to the one below x 7 Graph Fit Graphs 1 0 0 o 0 0 5 Concentration y A DIL ICI B 3 4 D B C D RAS B Hot Data Concentration vg 96 Activati 64 926 0 533 OOF 102 151 0 909 Figure 10 79 Cluster Bar Graph Displayed Step 10 Data Analysis Summary Formula SoftMax Pro User s Manual Scroll down to the bottom o
115. density or percent transmittance T within the reduction parameters of MinOD and MaxOD SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Lambda at Maximum Reports the wavelength at which maximum absorbance optical density or percent transmittance 76 T within the reduction parameters of MinOD and MaxOD Lambda at Minimum Reports the wavelength of minimum absorbance optical density or percent transmittance 76 T within the reduction parameters of MinOD and MaxOD Area Under Curve This reduction estimates the area under the curve as defined by the data plots in wells and cuvettes within the reduction limits The data plots are treated as a series of trapezoids with vertices at successive data points and at the X axis coordinates of the data points The areas defined by each of the trapezoids are then computed and summed Data Mode 96 Transmittance Absorbance For all read modes you can choose whether to view data as Transmittance or Absorbance OD As with all reduction or display options these choices do not affect the raw data acquired from the instrument but rather only how it is shown in SoftMax Pro If T is chosen all values including raw data are displayed in T AN KANTORA When the data mode is switched from T to Absorbance or vice versa SoftMax Pro recalculates all of the values based upon the new data mode Separate mathematical calculations are
116. example uses two controls with each control being placed in three wells The controls should be entered on the template as follows e Co01 wells A9 through C9 e Co02 wells D9 through F9 Start by highlighting wells A9 through C95 Select Controls from the Group menu The selected wells will now show the default first sample name for the Controls group Co01 Do the same for wells D9 through F9 assigning the default second sample name Co02 to these wells The template should look like the one below mi Experiment Plateiti seme o02 ml sees Print Cancel OF Figure 10 12 Controls Added 10 16 SoftMax Pro User s Manual Chapter 10 Tutorials H The last items to be added to the template are samples with dilution To do SoftMax Pro User s Manual this you will need to create a new group First choose the wells of the tem plate that will contain these samples A11 through C12 Click the Group pull down menu and choose New from the bottom of the list This causes a dialog box to appear allowing you to choose the attributes for the new group Name the group Sam w dil and choose Unknowns dilution for the Col umn Format This causes a checkmark to appear in the Sample Descriptor box and puts Dilution Factor into the text box automatically To the right of this text box define the units by clicking the arrow next to the drop down menu and choose ug ml The dialog box should loo
117. fit is y A B 1 exp x C a fate Fit Exponertiel ij ej Graphz B concentration y A B 1 exp xic yy A B c Ree Fiol iGroupz1 concentration vs Mean value 0 128 0175 28 984 0 363 Figure 8 48 Graph with Exponential Fit SoftMax Pro does not plot samples with descriptor values of zero 0 on a log axis Curve fits affected by this are the semi log log log log logit and 4 parameter curve fits SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Minimum Number of Standards The number of standards used in a standard curve depends on the particular curve fit selected and on the judgment of the operator The table below gives the minimum number of standards required mathematically by each of the curve fitting algorithms Table 8 5 Minimum Number of Standards Required for Curve Fits ter Qmm 3 site KENN mm 0 0 RO AINI NINJ N de 0 00 emer 000 4 Reemi 0000003 0 EL RN mem o miewl ny For optimal results you should always exceed the minimum number of stan dards required for any given fit There are other requirements on the standards for the log logit and 4 parame ter logistic curves The fits for the 4 parameter logistic curve are based on the assumption that the curve has a high and a low asymptote and a certain steepness for the linear portion around the inflection point between the a
118. fit is shown in Figure 7 46 w Z Graph t me Plots Fit Quadratic Y E Graph 1 O 5 oa 2 mE m m T i 15 Concentration 4 At Bx Cx2 a STD Standards Concentration vs Mean alue Z 18e 4 Figure 7 46 Graph with Quadratic Fit 7 58 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments SoftMax Pro User s Manual 4 Parameter Logistic and Log Logit If the standard curve has a sigmoidal shape when plotted on the semi log axes it may be appropriate to use either the log logit or 4 parameter fit Both fits are based on the equation E a x B La where D is the Y value corresponding to the asymptote i e the flat part of the curve at high values of the X axis and A is the Y value corresponding to the asymptote at low values of the X axis The coefficient C is the X value corre sponding to the midpoint between A and D The coefficient B describes how rapidly the curve makes its transition from the asymptotes in the center of the curve A large value of B describes a sharper transition Typically B has a magnitude of about 1 Both the log logit and 4 parameter equations are shown on the screen as y A D 14 x C B D O v Zl crape sw Ft 4 Parameter_v Graph concentration y CUR DII CXICIPB J O A B M D Fiti Groupz1 concentration vs Mean salue 0 064 0 779 11 314
119. for any or all read modes Endpoint Kinetic Spectrum or well scan Choosing Custom from any of the menus or dialog boxes causes a Formula button to appear an example of this button in an Endpoint Reduction dialog box is shown in Figure 7 26 Custom reduc tion formulas may contain other operators and terms in addition to wave lengths Reduction ro wavelength Combination poData Mode j Use plate blank rl Absorbance Formula e Transmittance Button Figure 7 26 Custom Selected Showing Formula Button Endpoint Clicking the Formula button opens the Calculation dialog box in which you can name and define a new reduction formula Calculation uu Accept Figure 7 27 Wavelength Calculation Dialog Box Endpoint Examples of some formulas that might be used when combining multiple wavelengths in the Calculation dialog box are given in Table 7 2 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Table 7 2 Wavelength Reduction Formula Examples Lim1 Lm2 Lm1 Lm2 Lm3 Lm1 Lm2 Lmn Lm1 Lm2 ILm1 Lim3 Lm2 ILm1 Lm6 Lm2 Lim5 Lm3 ILm3 Lm4 ILmx constant For example Lm1 1 44 for quantitating a polyclonal antibody b measuring the absorbance at A280 in a SpectraMax Plus or Plus with PathCheck on Average lallvalues This formula will average together the optical densities for multiple readings at
120. format to the file prefix e g Data 042136 Includ ing both date and time will create a file name that looks something like Data 8 14 01 042136 If you do not append the time and perform multiple runs either on the same day or with just a file prefix enabled SoftMax Pro will append an index to the file name starting with 1 and increasing by 1 with each successive reading Including the date and time ensure that the file will not be overwritten acci dentally by another autosaved data file Additionally appending the time may shorten the time required for Autosaving since the software will not have to search for the next number in the index series The recommended set ting for Autosaving files is to use both the date and time if all files are being saved to the same folder alternatively saving files to a separate folder for each day s runs may be useful When Autosave is enabled in SoftMax Pro file mode for files con taining multiple experiment sections each experiment section will be autosaved to a separate file If you are using SoftMax Pro with ROBOmax you must deselect the Autosave and Autoprint functions ROBOmax software automati cally saves the SoftMax Pro data file after the plate is read SoftMax Pro User s Manual 1 3 Chapter 9 File Management and Printing Printing Five printing options are available for an experiment 1 You can print a report at any time by choosing Print 3 P CT
121. groups and indicate the type of samples the wells will contain Depending upon the default protocol you are using certain group names and associated sample descriptions may have been created You can use these predefined groups and or create new groups to suit your needs When creating new groups you may enter any name you wish Once entered this name will be available for use in that experiment thereafter New groups can be created independently from selecting or assigning any wells in the Tem plate Editor Each well designated as being part of a group has associated with it a group name a sample name or replicate ID a sample descriptor optional and a default column format group type such as Standards for example When a group is applied to selected wells the group name is displayed in bold type over the wells and the well bear the same color 5 19 Chapter 5 Reading Using Fluorescence or Luminescence Instruments 5 20 Template Editor Tool Bar Group Drop Down List Microplate Map Sample Descriptor Field Clear Edit Sample Series Assign Button Button Drop Down List Button Button Experimentz 1 Ph mno X Group Standards w e alb CNN 7 Series les f Assign Clear A Concentrations Soe Se pum Figure 5 17 Template Editor Dialog Box Groups are always visible both in the Template and in the data display of the Plate section Even though
122. hard disk in a different folder or subdirectory Saving Files under the Same Name If you have made changes to a file and want to save these changes thereby updating the same file select Save 3 5 CTRL S or Save As from the File menu If you select Save 3 5 CTRL S the current version of the file will replace the old one without notification If you select Save As an alert box will appear asking if you want to replace the previous file by that name If you are intentionally replacing the file click Replace or Yes The new information will be written over the old information under the same file name Saving Files under a Different Name or in a Different Folder If you have made changes to a file and want to save these changes as a new file or if you wish to save the file to a different location select Save As from the File menu A dialog box will open allowing you to enter a new name SoftMax Pro User s Manual meu Chapter 9 File Management and Printing Autosave SoftMax Pro User s Manual and or a new location for the file To choose a different location for the file use the pull down menu at the top of the dialog box to select a different folder To change the name overtype the existing file name Click when you have entered all the necessary information and you are ready to save the file SoftMax Pro limits the number of characters that can be used in a file name On the Macintosh file names ar
123. have been defined the sample menu will look like the one below The check mark indicates the sample that is currently being named Saroiple Sta0z ul Concentration lo mq n Figure 4 25 Sample Drop Down List Sample Names Defined and Wells Selected Sample Descriptor Field The field associated with the sample descriptor only appears in the Template Editor tool bar if the group column type that has been selected requires this type of information Generally the information entered in this field is a dilu tion or concentration Position the cursor in this field and enter a number The label and units assigned to the sample descriptor in the Group Settings dialog box are shown on either side of the field Assign Area SoftMax Pro User s Manual The Assign area contains the Series and Assign buttons Figure 4 26 Assign Area of the Template Editor Tool Bar Assign Button and Function Click this button to apply a sample name to one or more wells that have been selected in the template You may also perform this operation by using the Enter key on your keyboard 4 29 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments k Series Button and Function This button is used to define several samples as a series allowing you to easily enter incremental sample descriptors for example dilutions or concentra tions and sample names to the template as long as the increment can be express
124. in 1000 s Cancel w Display E Plot RFU RLU h vs ue Wavelength With reduced number default Cancel z Raw Reduced nm Reduced Display Choices Lb v Numb Threshold Ranged Grayscale Cancel mw Display 2 Raw Reduced Ymax units per sec Lm1 Example Options BEE n Reduced Threshol Ranged Display pre Grayscale c s Choices Cancel w Display Py Raw Reduced Raw RFU Example Raw Data Plot RFU RLU vs Time default Cancel ms Display Show With reduced number Raw Reduced Raw RFU Example Average RFU RLU per Well Show With reduced number Raw Data in 1000 s s Cancel mi Display E X EX Raw Reduced ReRU ld Example Raw Data Plot RFU RLU vs Ln Time with reduced number default Cancel SoftMax Pro User s Manual ms Display Z Raw Reduced Lambda at Maximum Lrm1 Example Options Ux wv Number Dew Reduced Displa Grayscale 1 Plot F y Choices Cancel ms Display X Raw Reduced Average ILm1 Example ike wv Number Threshold Reduced Display Choices Ranged Grayscale Plot Cancel mi Dispay xj Xx Raw Reduced VMax units per sec ILm1 Options n ux wv Number Reduced
125. is greater than or equal to the calculation of MinStd found in the Standards group section and less than or equal to the calculation of MaxStd found in the Standards Group section then show nothing signified by two quotation marks with nothing between them otherwise show the word Outlier Looking at the Standards section you will not immediately see the MinStd or MaxStd calculations used by this If statement They are there but their names are hidden Look at the bottom of the Standards section and you will see two summaries as shown in the following figure SoftMax Pro User s Manual 10 29 Chapter 10 Tutorials 10 30 Lu Ea fen E Standards ugiml Figure 10 35 Summaries in the Standards Section Double click the first summary Smallest standard value A Calculation dialog box will appear W Calculation Description Smallest standard value Formula MinthWean value Decimal Places Agent Cancel Figure 10 36 Calculation Dialog Box from Summary in Standards Section This dialog box contains a checkbox for Hide Name which is currently enabled Unchecking the box will allow you to view the name of the sum mary to the left of its text in the Standards section The formulas for MinStd and MaxStd are Min Mean Value Max Mean Value The MeanValue column is derived from the average of the well values found in the Values column the Standard replicates The minimu
126. maximum rate SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Onset Time This is another method for analyzing non linear Kinetic reactions Onset Time reports the time required for a Kinetic reaction to change by a specified opti cal density onset OD above the initial OD reading for the well This elapsed time data is useful for cascade reactions including clot formation endotoxin testing for example and clot lysis applications where the change in reagent concentration does not affect the maximum optical density change but changes the time required for the reaction to reach completion Time at Minimum This setting reports the time at the minimum OD or T that falls within the reduction limits Time at Maximum This setting reports the time at the maximum OD or T that falls within the reduction limits Time at 1 2 Maximum This setting reports the time at the half of the maximum OD or T that falls within the reduction limits To calculate this reduction SoftMax Pro first determines the Kinetic point within the reduction limits that has the maxi mum signal level OD or T It then scans the Kinetic plot from left to right until it finds two points having signals that bracket half of that value A linear interpolation between these two points is used to estimate the Time at 1 2 Maximum Slope This setting determines the slope of the combined plot i e the slope of th
127. menus The Graph Options Dialog Box The Graph Options dialog box allows you to change the name of the Graph section change the height of a graph select the type of graph to be displayed change the fonts used on the title legend and axes access the Plot X dialog box for editing plots entering new plots and or to delete plots This dialog box is opened after pressing OK in the Plot X dialog box when a new Graph section is being created This dialog can also be accessed by pressing the Graph Options button ft on the Graph section toolbar or by choosing Graph Options from the Graph menu Scatter plots cluster bar charts and stack bar charts can be created Error bars can be used with all graph types You can also choose whether to connect the points and or plot the symbols in the graph Each of the plots that have been created for this Graph section are listed in the Plots field of this dialog box Choosing any of these plots provides an indi cation of the data to be used for the X and Y axis data points and error bars on the right side of this field Plots can be deleted by highlighting the plot name and clicking the Delete button Clicking the New button opens the Plot X dialog box allowing you to create a new plot for the graph Existing plots can be edited by highlighting the plot name and clicking the but ton which opens the Plot X dialog box containing all the information for the selected plot When crea
128. next to Before First Read to shake the microplate before the first reading Whether you read at a single wavelength or at multiple wavelengths enabling Auto mix for Before First Read will shake the plate for the set amount of time before the first wavelength reading only You can accept the default value shown for the amount of time to shake the plate or enter a different value up to 999 seconds in the box to the right Between Reads This option is applicable only to Kinetic readings and will be displayed only when Kinetic mode is selected To enable automatic mixing of the microplate between readings during a Kinetic assay click the checkbox next to this set ting For readings at a single wavelength Between Reads will shake the plate for the set amount of time prior to each reading at that wavelength For readings at multiple wavelengths enabling Between Reads will shake the plate for the set amount of time before each reading at the first wavelength only You can accept the default value shown for the amount of time to shake the plate or enter a different value up to 999 seconds in the box to the right SoftMax Pro User s Manual 5 17 Chapter 5 Reading Using Fluorescence or Luminescence Instruments AutoCalibrate This checkbox allows you to disable or enable automatic calibration The default setting is checked enabled Turning AutoCalibrate off allows the instrument to complete readings more quickl
129. not affect the exported data since only the raw number is exported when raw numbers are displayed and only the reduced number will be exported when reduced data is displayed 5 Choices made in the Export dialog box After choosing Export from the File menu a dialog box will appear asking you to choose which sections you wish to export You can choose to export Plate and or CuvetteSet sections all or selected and or Group sections all or selected After choosing a name for the exported data file click to save the exported data SoftMax Pro User s Manual 9 17 Chapter 9 File Management and Printing Export Data to File name A Selected BLOCKS 1 Plate Plate 1 1 PlateFormat Endpoint Absorbance Raw FALSE 1 300 9 350 750 0 1 0 11052 0 12214 0 13499 0 14918 0 16487 0 18221 0 20138 0 22255 0 24596 0 27183 0 30042 20 80 0 12214 0 14918 0 18221 0 22255 0 27183 0 33201 0 40552 End 0 13499 0 16487 0 20138 0 24596 0 30042 0 36693 0 44817 0 14918 0 18221 0 22255 0 27183 0 33201 0 40552 0 4953 Save in cy SOFT mas PRO hd Group Sections i All C Selected Untited txt save as bype Test Files tst ad Cancel w Plates and Cuvettes i 6 30 captures cr Additions3 DOC iL Assays ey Changes to Chapter 6 D0C Export Data to Untitled Text ff Plates and Cuvettes C3 All i amp Selected Cancel Group Sections C All g
130. of Group sec Opens the Group settings Standard tion dialog box double click Group Settings in the Opens the Group Settings Group menu dialog box Opens the Calculation dia log box allowing you to create a summary FH double click Create Summary in the Group menu Opens the Calculation dia log box allowing you to create a column Create Column in the Group menu Edit Summary in the Group menu or dou Opens the Calculation dia ble click the summary log box allowing you to pointer changes to edit the summary double headed arrow Edit Column in the Opens the Calculation dia Group menu or dou log box allowing you to ble click the column edit the column Shows hides the formulas used in columns and sum maries Show Hide Formulas in the Group menu Enlarges or reduces col umn size to fit the largest piece of text Autosize in the Group menu Hides one or more selected columns or shows all col umns that were hidden Hide Replicates in the Hides data from all repli Group menu cates i Includes excludes section Printer icon from printed report Hide Show All in the Group menu D 3 20 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Graph Section Tool Bar Body Plot Information SoftMax Pro User s Manual xz 7 Standard Curve Fit T 1000 Standard Curve Logi B Logix O Plaot 1 Standards Concentration vs PTean alue Fig
131. of the logarithm of the readings on the Y axis the response and the logarithm of the dose on the X axis The resulting display will be a straight line with both axes drawn in logarithmic scale The equation for the log log fit is log4o Y A t B log49 X and is shown on the screen as log y A B log x where A is the log Y intercept of the line for log49 X 0 and B is the slope note log in this equation is the common or base 10 logarithm E StandardCurve Fit LogzLog StandardCurve 1 2 a p m Cc wm 2 Concentration LoglyJ amp B Logix o STD Standard Concentration vs PTeanValue Figure 8 42 Graph with Log Log Fit SoftMax Pro User s Manual 6 55 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments l k Quadratic The quadratic function fits the best parabola to the data The parabola is a curved line based on the equation y A4 B x C x which is shown on the screen as ysASXBx4C2 where A is the intercept B is the slope of the curve at the intercept and C is the measure of the curvature of the parabola The quadratic fit is most appropriate when the standard curve has a tendency to curve up or down An example of a quadratic fit is shown in Figure 8 43 w Z Graph t me Plots Fit Quadratic Y E Graph 1 O 5 oa 2 mE m m T i 15 Concentration 4 At Bx Cx2
132. open a file To perform this sec ond option do the following In My Computer or Windows Explorer click the View menu and then click Options Click the File Types tab In the list of file types click Printshop The settings for Printshop will be shown in the File Type Details box PDA will appear as the extension used Click Edi t In the actions box click Open Click Edit and then specify SoftMax Pro as the program you want to open files that have this extension To do this click the Browse button and locate the SoftMax Pro executable file Doing this will cause pda files to be associated with SoftMax Pro instead of Printshop In order to open Printshop files you will need to open the Print shop application and open pda files relating to Printshop from within that application double clicking a pda file will open SoftMax Pro SoftMax Pro User s Manual Chapter 12 SoftMax Pro Remote Command Language In EO QW CLIO EL e o2o daret aicut pil e bore emo ae tare ae oO 12 3 Notes On Queued Processing 0 0000 essen 12 3 Send Receive Remote Commands 0 22 ee eee eres 12 4 Summarizing Remote Commanding 0 12 7 Programmer NOLS resinen nehan o ber eta ee bon Rune ide aon 12 7 Molecular Devices Position iov REC EESE REY 12 8 Soft Max Pro Commands 94d t ee PET aces uer eruta won he eee d 12 9 Visual Basic Excel Macro Example sse 12 1
133. pR Average Values InterpX STD Stan dardCurve Values Average Result Stdev Result Cv Result Formula Name Mean Value Formula Average Values Name Formula Stdev Values Name CV Formula Cv Values Name Formula Name Formula Name Formula Unknown Dilution Sample ISampleNames Wells WellIDs Values WellValues R Outside Standard Range If Values MinStd Standards and Values lt MaxStd Standardsy 44 7 RR J Result InterpX STD Standard Curve Values Meankesult Average Result Std Dev Stdev Result CV Cv Result Dilution Factor Adj Result MeanResult Factor Concentration and Factor are from SoftMax Pro version 1 x Concentra tion Factor and SampleDescriptor return identical information A No Ox SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments SoftMax Pro User s Manual The Button When you click the button the Group Settings dialog box will appear reflecting the settings for the active group currently visible in the group drop down list If you wish to edit a group that is not currently active first choose the group from the drop down list and then click the button The fig ure below shows the Group Settings dialog box for the active group called Patient 1 broup Settings Mame Patient 1 Name of Group Sample Descriptor VEJ Sample D
134. permissions defined are Lab Assistant user group with permission to change instrument settings and read plate only Method Creator user group with required permissions to create proto cols Data Analysis user group with permissions to change reduction settings Approval user group with permission to sign e statements After determining the assignment of permissions you will be ready to add users to the Enterprise Administrator database You may find it useful to make copies of the following sheet to record permis sions for the users in your group s Installation NOH Enterprise Administrator is available only for Windows based com puters hey If you are not the administrator no installation of Enterprise Administrator is necessary Follow these instructions to install Enterprise Administrator 1 Insert the Enterprise Administrator CD into the CD ROM drive SoftMax Pro User s Manual 6 7 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator 2 3 4 5 Open My Computer and then double click the CD ROM drive icon to display its contents Double click the Setup icon and follow the screen prompts The Welcome screen suggests that you close all other programs before begin ning installation of Enterprise Administrator software It is also recommended that you disable any virus protection software you may have running before installing Enterprise Administrator Click Cance
135. plot Kinetic data from well A1 in a Graph sec tion you can enter the formula A1Lm1 for the Y Axis and the TimeRun accessor for the X axis The entered formulas are also shown in the Source field of this dialog box The size color and style of the icon used for a plot can be selected on the right side of this dialog box Four sizes nine colors and nine styles are available from drop down menus The Graph Options Dialog Box The Graph Options dialog box allows you to change the name of the Graph section change the height of a graph select the type of graph to be displayed change the fonts used on the title legend and axes access the Plot X dialog box for editing plots entering new plots and or to delete plots This dialog box is opened after pressing OK in the Plot X dialog box when a new Graph section is being created This dialog can also be accessed by pressing the Graph Options button ft on the Graph section toolbar or by choosing Graph Options from the Graph menu Scatter plots cluster bar charts and stack bar charts can be created Error bars can be used with all graph types You can also choose whether to connect the points and or plot the symbols in the graph Each of the plots that have been created for the Graph section are listed Choosing any of these plots shows the data that will be used for the X and Y axis data points and whether or not error bars will be used Plots can be deleted by highlighting th
136. privileges can be created to make assignment to users easier e Report The administrator may export user information and privileges to a text file and view an audit trail of administrator activities e License The administrator may add new licensing information to Enter prise Administrator This information will be used by Enterprise Adminis trator to control the number of users registered to use Molecular Devices software programs The person chosen to administer this program may also set certain global options to make Molecular Devices software more secure these options are described on page 6 14 How Enterprise Administrator and the Enterprise Edition of SoftMax Pro Work Together SoftMax Pro User s Manual e The administrator installs Enterprise Administrator software Windows only on his her PC The administrator adds SoftMax Pro Enterprise Edi tion user names to Enterprise Administrator to create a user database Each user is assigned permissions that determine the activities that will be avail able that user within SoftMax Pro e The administrator then installs SoftMax Pro Enterprise Edition Mac or Windows on individual PCs belonging to users or set up with an instru ment and connects SoftMax Pro to the Enterprise Administrator database e A Guest a user not in the database can open and view files and can print those files The Guest privilege facilitates FDA review of a file if needed The following privilege
137. processing is not enabled this command does nothing Close the current document NISNUNNBH If the data has not been saved the document is closed any way with no user warning CloseCDrawer FlexStation only Tell SoftMax Pro to close the compound drawer CloseDrawer Tell SoftMax Pro to close the instrument drawer CloseTDrawer Copy FlexStation only Tell SoftMax Pro to close the tips drawer This is equivalent to pressing CTRL C or selecting Copy from the Edit menu Notes e If a plate section has just been read or is the only selected section the plate data will be copied to the clipboard e The data copied will follow the display settings for the current plate nor mally Raw Data Other sections such as Groups can also be copied The SelectAll command should be used first ExportAs X XX SoftMax Pro User s Manual Tell SoftMax Pro to export data in the standard SoftMax Pro format e Attribute available a The ExportAs command without the a attribute overwrites any existing file without warning However you can choose to append export data to the 12 9 Chapter 12 SoftMax Pro Remote Command Language 1 B 12 10 end of an existing file by specifying the a attribute In addition this exported file can then be imported and all data can be viewed as a single doc ument file Please note that when using the attribute a a single space is required between the attribut
138. section tool bar This selection is valid only with data shown in plots format Well Scan SpectraMax Gemini Gemini XS and FlexStation only Some applications involve the detection of whole cells in large area tissue cul ture plates Well Scan mode can be used with such microplates to allow maxi mum surface area detection in whole cell assays Since many cell lines tend to grow as clumps or in the corners of microplate wells you can choose from several patterns and define the number of points to be scanned to work best with your particular application Zoom Box Macintosh A small box containing a smaller box at the right of the title bar You can click the Zoom box to return the window to its previous size B 16 SoftMax Pro User s Manual Index Symbols Baseline with Multiplier 8 34 Transmittance 1 4 4 9 4 11 4 16 7 12 7 18 7 24 7 36 7 37 9 20 B 5 B 13 B 15 Numerics 1 Ref 7 39 4 Parameter curve fit 7 59 8 57 A About SOFTmax PRO 3 26 Absolute values 7 18 7 31 7 32 8 24 8 25 8 28 8 34 Absorbance 1 3 1 4 4 11 4 16 7 18 7 24 7 37 B 1 B 5 B 10 B 13 Active 3 9 3 10 3 11 3 24 3 26 3 27 3 28 3 30 3 34 B 1 Add Software Enterprise Administrator 6 19 Add Statements 6 29 Administrator 6 5 Administrator Login 6 8 Alerts 11 3 ANSI Character Set B 1 AppleTalk 2 4 11 12 11 13 Area Under Curve 4 10 5 11 7 33 7 35 7 36 7 37 8 26 8 28 8 29 8 30 8 33 B 1 ASCII C
139. see eo seer DEP INE Td dde ded Uto qdudd 9s eases 7 28 Spectrum SpectraMax Instruments Only sss 7 36 Data Mode Transmittance Absorbance 7 37 Custom Reduction Formulas 0 0 0 0 eee eee 7 38 Recalculation Options 00 0 0 7 40 Displaying Data in Group Graph and Notes Sections 7 41 CHOU DECOR Dur oad reed Sq POS OPE Sore taped aab 7 41 INO1GS C UON P 7 47 euren T M 7 47 Formulas in SoftMax Pro een eee 7 67 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments cue a Ra REP EER TEES 8 1 OG BEC HOTELS ico pon ep marina da wu e nO Tae v res WES Nd xd RS d do dd 8 3 Initial Data Display and Reduction 00 0 0 eee 8 4 The Data Dipli R 8 5 The Display Dialog Box leeeseeeeeeeeeeeees 8 6 Craphine Welle e eorepd cobre dep uw Sd addi d x dads 8 14 Mackine Wele EPUM 8 18 Ate Keda O eera T E E diri ER EEEE vw oe 8 19 FAE mp AEE A EAE R ASN 8 21 E EE E E EEE EEEE 8 21 DDOCULHII ea ee a EPI ET A ra ended es ad 8 29 Wel SClts4 era RE EEEE E 8 30 ETA Cr ne E ee E E E ee ee eee 8 31 Custom Reduction Formulas 0 00 00 essen 8 35 Recalculation Options o rerrreeniireriereri resti yiti niis 8 37 Displaying Data in Group Graph and Notes Sections 8 38 rP CO 4 943 ded E RER s 8 38 NOCE CCOO err yeer E PUES Haus Ey Awd nau E ip xe 8 45 Graph SeCHON 2iencctetoute NIS OU gabaetesecustar
140. setting for more precision with a greater number of readings per well The default number of readings per well varies with the read type for fluores cence the default is 6 for luminescence the default is 30 for time resolved flu orescence the default is 20 The number of readings is variable from 1 to 30 With the luminescence read mode the integration time is directly proportional to the number of readings per well determined by this setting Each reading per well is equal to 3 2 milliseconds of integra tion time 5 42 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments PMT Sensitivity The PMT photomultiplier tube is a vacuum tube that detects light espe cially from dim sources through the use of photoemission and successive instances of secondary emission to produce enough electrons to generate a useful current Sensitivity r Readings Fast Figure 5 40 PMT Sensitivity Setting Clicking in the box causes a menu of choices to appear Settings are manual and limited to High Medium or Low The voltage of the photomultiplier tube may be set to low for samples having a higher concentration medium or high samples having lower concentra tion in all read modes AutoCalibrate This checkbox allows you to disable or enable automatic calibration The default setting is checked enabled Turning Autocalibrate off allows the instrument to complete readings more quic
141. settings do not affect the raw data but simply cause it to be dis played differently You can change reduction settings without affecting the raw data from the plate or cuvette reading in any way Custom reduction formulas require the use of accessors and operators that are understood by SoftMax Pro A list of these accessors and operators is given at the end of this chapter 41 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments l B Resizing and Hiding Columns The columns in a Group section can be resized in several ways One of the simplest methods is to drag the line dividing the columns to another position moving it horizontally When you point to a line between columns the cur sor changes to a double arrow with a vertical line through it showing that it is ready to move left or right see Figure 7 28 Double headed Std Dev CV arrow Cursor Smallest standard value Largest standard value Figure 7 28 Cursor Positioned on a Line between Columns Reducing Column Size and Hiding Columns You can reduce a column to a size smaller than the width of its title in which case the title will not be completely visible You can also hide a column by reducing its size completely drag one edge of the column on top of the other or by selecting that column and choosing Hide from the Group menu If you hide a column in this way and then wish to view it again use the Show All command in the Group menu
142. should be cautious about using correlation coefficients as an indicator of goodness of fit Do not let this single value for goodness of fit sway your intuition about which fit is best Some curve fits may seemingly give a good fit e g fit values close to 1 but when inspected by eye show a poor fit along a major portion of the plot Graph Options Graphs can be customized further through the Graph Options dialog box which is accessed by choosing Graph Type from the Graph menu or by click ing the Graph button Jin the section tool bar Graph Options Title Graph Height Type SU Scatter Sample Text m Mh 3 Cluster bar Sample Text Set hl Stack bar Legend no Connect points 5 le Text nen Sets Flot symbols Flots Mew O Plot 1 O Plat 2 gt Std Dey eStandards Y MaluesmStandards A Errorz Wo Error Y Errors Mo Error Figure 8 49 Graph Options Dialog Box The default options are shown in Figure 8 49 but you can alter these as desired you may change the font and type size used for the title legend and or axes of the graph and you can choose the color and type of symbol used on a scatter graph to show the plot The graphs shown in preceding Fig ures reflect the default graph options Connecting the points of a scatter graph can be useful in determining the closeness of the fit Figure 8 50 below shows a scatter graph with points connected compared to a linear fit 99831 The Connect P
143. starting value choose the Step by function and enter O 25 for the increment of the series These choices will cause the series of wells you have selected to be assigned concentrations of 0 25 0 5 0 75 and 1 0 ug ml respectively The choices you have made should look like this SoftMax Pro User s Manual 10 13 Chapter 10 Tutorials l 3 Start From First Sample t03 i i Top che v Sample Descriptor mmm 2 Bottom Concentration Units mE E64 Left Starting value comp 7 Right Step by 7 TEN Replicates Cancel Figure 10 7 Series Dialog Box Click the button to close the dialog box and return to the Template Edi tor The four sets of wells you selected should now be assigned the values entered for the series You can check the concentrations assigned to these wells by pressing and holding the CTRL Shift keys m Expermentit1 Plate 1 Eat Sample a ms so vl senes le Shoup Series zamn Clear Concentration 025 pon 1 d d 4 J 12 EN m D LOIRE Cancel OF Figure 10 8 Template with CTRL Shift Keys Depressed E Thelast standards need to be entered manually Select wells H1 and H2 A new sample descriptor is created automatically St07 so all you need to do is assign a concentration value of 1 4 ug ml to these wells After entering the value click the Assign box or press the Tab or Return key to assign this value to the selected wells
144. the Instrument Settings Dialog Box for Absorbance Instruments 4 504 54s berba 99 dota dr edad Donat E Poeti krone 4 5 Columns Created and Their Formulas 0 cece eee cece eee eens 4 26 Column vs Wavelength Priority 0 0 eee ccc eee ee 4 45 Features Available in the Instrument Settings Dialog Box for Fiuerescence Instruments aene medie dictos a 004 9 8 ROS H Ap o ie cionis senses anne 5 5 Columns Created and Their Formulas ssleeeeeeeeeeeeeeee 5 24 Column vs Wavelength Priority 00 cece eee 5 36 Using Vmax Points Example see eruit cud batum pd aae deeds a RUPU da nes 7 34 Wavelength Reduction Formula Examples 0 eee e eee eee eee eee 7 39 Kinetic and Spectrum Reduction Formula Examples 00 00 005 7 40 Minimum Number of Standards Required for Curve Fits 005 7 64 Using Vmax Points Example 24520202202 tates S94 9d d EU ede dud da 8 27 Using Vmax Points Example sacs 24 eed eso donee A eiue dunt 8 31 Wavelength Reduction Formula Examples 00 cece eee eee ee eee 8 36 Kinetic and Spectrum Reduction Formula Examples 0000005 8 37 Minimum Number of Standards Required for Curve Fits 005 8 61 PC File Name Extensions and Macintosh Document Types 2 005 9 7 ASCII Data File Fields and Descriptors llle 9 20 ASCII Template File Columns and Descriptions 00 0000 cec
145. the Plate section the data display will show the group areas in different colors Button Equivalent Template in the Plate section tool bar Reduction Opens the Reduction dialog box Options available in the Re duction dialog box depend on the type of reading performed End point Kinetic Spectrum Well Scan or FLEX and the instrument used to collect the data The Reduction dialog box is used for initial reduc tion of the data collected from the microplate reader The optical den sity information collected is reduced to a single number per well More information regarding reduction options can be found in Chap ters 7 or 8 choose the chapter that is appropriate for the instrument you are using Button Equivalent in the Plate section tool bar Display Opens the Display dialog box Allows you to decide how the infor mation in the Plate section will be displayed More information re garding display options can be found in Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using Button Equivalent in the Plate section tool bar Graph The Well Graph command is modal so it can be activated both dur ing data acquisition and after data has been acquired for a Kinetic Spectrum Well Scan or FLEX reading You can get a close up view of the plot shown in one or more wells by first selecting the well s and then choosing the Graph command This command is dimmed if the instrumen
146. the column or summary and then choosing Edit and then Text Style Ctrl T you can change the font size and or style of the text Notes Sections The Notes section allows you to enter text describing the experiment and one or more summary lines that contain formulas To create a summary click the Create Summary button in the tool bar or choose Create Summary from the Notes menu The Calculation dialog box that appears see Figure 7 34 allows you to enter a description of the summary accept the default title for the summary change it or hide it and enter a formula including the number of decimal places the reduction should show if appropriate Detailed information regarding oper ators and accessors can be found in the Formula Reference Guide If you wish to see the formulas associated with the summaries without calling up the Calculation dialog box for each one you can choose Show Formulas from the Notes menu To see these formulas only briefly hold down the shift and control Ctrl keys while that Notes section is active The summaries will expand to show the complete formula along with the other summary infor mation They will resume their previous appearance when you release the option key Graph Section The Plot X Dialog Box SoftMax Pro User s Manual Whenever you create a new Graph section or add a plot to an existing graph the Plot X dialog box opens automatically X starts with 1 and incre ment
147. the loca tion where saved files will be stored and the autonaming conven tions that will be used Autosave e Whether or not reports should be printed automatically and print settings that will be used Autoprint e Wavelengths of filters installed in the Emax Vmax UVmax or ThermoMax instruments not used with SpectraMax FlexStation or VersaMax instruments Suspend Recalculation Stops SoftMax Pro from performing calculations based on changes you make to the settings or other program informa tion This command is used most often when you want to make a number of changes to the settings without having to wait while the re sults of each change are recalculated Recalculate Now CTRL 2 Causes SoftMax Pro to recalculate data based on the current settings This command is used most often when you have suspended automatic recalculation SoftMax Pro User s Manual 3 29 Chapter 3 SoftMax Pro Overview 1 F View Menu The View menu provides three commands Minimize 6M CTRL M Expand 32E CTRL E and Hide Status In addition to these choices the names of the experiments appear at the bottom of the menu sections within experiments are shown as submenus allowing you to go to a specific experiment or sec tion within an experiment by choosing the item If the section was closed selecting it from the View menu will open and activate it The currently active section is shown in the menu with a checkmark next to it
148. the maxi mum signal e Well Scan Fluorescence data is collected from a single well in a micro plate using one of several scan patterns up to a maximum number of points Data is displayed in gray scale by default readings from multiple wave length pairs can be shown separately or together if together they are dis played in different colors e FLEX FlexStation only Fluorescence data is collected from a single well in a microplate over time Readings from multiple wavelength pairs can be shown separately or together if together they are displayed in different colors A chart showing the read modes that are available for absorbance instruments can be found in Table 4 1 on page 4 5 a chart showing the read modes that are available for fluorescence luminescence instruments can be found in Table 5 1 on page 5 5 ose For absorbance instruments any of the read modes can display data as Transmittance or OD SoftMax Pro can collect data from one or more microplates and or cuvettes and store it in a single data file using the same or different instrument set tings for different microplates or cuvettes For example microplates contain ing different samples can be read using the same or different modes all within the same experiment New to SoftMax Pro If you have not used SoftMax Pro before Molecular Devices recommends that you install the software and then proceed to Chapter 10 Tutorials which guides you through
149. the name will still show in the group pull down menu and the Group sec tion will still exist SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual Group Settings Name Sample Descriptor Musei Rl ER wee cR e RR ER RR E RU DB RC ER T RR e Concentration units rnl Figure 5 20 Group Settings Dialog Box Using Default Settings Name Use either the default name Group X where X is an integer that is initially set to 1 and increments i e 1 2 3 etc as other groups are created or enter a custom name up to 32 characters in length In the Sample Descriptor area of the Group Settings dialog box you can enable the application of the sample descriptor if appropriate by clicking the checkbox You may also choose the units used for the sample descriptor by selecting an option from the Units drop down list or by typing the units you wish to use into the list box The Sample Descriptor is automatically enabled when either the Standards or Unknowns Dilution column format see below is selected The descriptor defaults to Concentration with the Standards column format and to Dilution with the Unknowns Dilution column format The Sample Descriptor may also be used for time or fraction number information No sample descriptor is assigned automatically for the Basic or Unknowns column format You may assign a sample descriptor to the
150. the same wavelength for example if you read the well six times at 280 nm Min lallvalues This formula will report the minimum OD recorded for multiple wavelength readings in each well Max lallvalues This formula will report the maximum OD recorded for multiple wavelength readings in each well If Lmz A makeerr 118 if L This formula will report low for any well with an OD less than A mx gt B makeerr 117 Lm1 high for any OD greater than B and the OD of any well that lies between A and B For Emax Vmax UVmax or ThermoMax instruments the choice available for reading at two wavelengths is designated 1 Ref While the instrument performs a reading at the two wavelengths you desig nate one for the measurement and one for the reference data regarding these individual wavelength readings is automatically reduced to a single value in the data display the value obtained from the reading at the reference wavelength is subtracted from the read ing at the measurement wavelength and only the resulting value is displayed When you choose 1 Ref you cannot view the data associ ated with the readings at the individual measurement or reference wavelengths SoftMax Pro User s Manual 7 39 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments l B Custom reduction formulas utilizing mathematical operators or terms can be used to obtain specific types of data Table 7 3 provides some examples of s
151. time to be shorter than the instrument s minimum read interval the program will alert you with a warning message An interval that is too short will cause a warning message to appear telling you that no calibration can occur between reads The minimum read interval is determined by the instrument that is connected to the computer or if no instrument is connected by which instrument has been chosen in the Reader section of the Preferences dialog box and the par ticular settings chosen for that instrument The minimum interval depends on the number of wavelengths the Automix status the wells to read and the distance the monochromator and filter wheel of the instrument must move If you are writing a protocol that will be used with an instrument other than the one connected to the computer or chosen in the Reader preferences you can disregard the alert and save the setup However if you attempt a reading using a protocol which has settings that are 5 11 Chapter 5 Reading Using Fluorescence or Luminescence Instruments not correct for the particular instrument you are using an error mes sage will instruct you to change the setting that is out of range before you can initiate a reading NOFA To achieve the shortest possible interval for Kinetic readings using the SpectraMax Gemini fluorescence readers or FlexStation instru ments choose wavelengths in ascending order Spectrum Measures relative fluorescence or luminescence uni
152. to define groups and assign wells within the Template Editor first The values received from the MDC Microplate System instrument are raw absorbance readings and are not affected by settings in the Template Editor Only instrument settings must be defined prior to reading a microplate To read a microplate open the drawer of the MDC Microplate System instru ment click the drawer button in the status bar or choose Open Drawer from the Control menu and insert the prepared microplate Then simply click the Read button in the status bar or choose Read from the Command menu If you have created more than one Plate or CuvetteSet section and no Plate or CuvetteSet section is active choosing the Read command will cause a dialog box to appear asking you to choose which section to read see Figure 4 37 Select the correct section and then click to close the dialog box If a Plate or CuvetteSet section is active choosing the Read command will read that plate or cuvette set Select Section elect Section g Sections Plate l w Bifsections Plate 1 p Plate 2 CuvetteSet l Figure 4 37 Select Section Dialog Box The screen on the right shows the choices available when two Plate sections and one CuvetteSet section have been created in one experiment A similar dialog box will appear if you have chosen to pre read the plate for blanking At the bottom of this dialog box however are two option radio buttons allowing you to proceed
153. to load anything in the actual instrument Instead choose Open from the File menu and navigate to the Tutorial folder Open the file named Tutorial2 pda The data in this file was generated using the parameters you have just created in this protocol file After clicking the button values would begin to appear in the data display in the Plate section as they were read by the instrument The plate section of the Tutorial2 pda file should look like the figure below v E nar sepu A E hax Min Plate 1 FLEX Time 200 secs Interval 2 secs Reads 104 Ex Em Cutott Lind 485 525 515 Atomix Off Calibrate On PMT High Reads Vell 3 Lag Time 0 End Time 200 RFU Min O RFU Max S0000 Wavelength Combination Lrn1 Checksum Failed Plate Last Read 3 17 PR 124 2 2001 Figure 10 62 Plate Section with Data from Saved File The display for each well that is defined by a group boundary shows a raw value with a reduced number The wavelength reduction formula is shown at the bottom of the display Wavelength Combination Lm1 the kinetic reduction formula is shown at the top of the Plate section Max Min Click the Display button in the Plate section tool bar The Display dialog box will open SoftMax Pro User s Manual t SoftMax Pro User s Manual Chapter 10 Tutorials Display Raw Raw EFU m With reduced number Figure 10 63 Display Dialog Box The current display selectio
154. to read a single well in Gemini and Gemini XS Enhanced 4 Parameter Fit e The 4 Parameter curve fit has been enhanced to be more robust Documentation amp Software Enhancements e A robotics program example has been added to the CD files provided e Added examples to the Formula Reference Guide The results of these exam ples are included as PDA files that can be used as tutorials Life Cycle Documentation for SoftMax Pro 4 0 is included in the SoftMax Pro 4 0 Validation Manual e A feature request plan and testing plan are included in the SoftMax Pro 4 0 Validation Manual Specifications e Added support for Windows ME e Dropped support for Windows 95 and Mac OS below 8 6 A 10 SoftMax Pro User s Manual Appendix A SoftMax Pro Version Specifications e Increased minimum required computer hardware and software specifica tions when FlexStation and or robotics are used SoftMax Pro 4 1 Specification Specifications Added support for the SpectraMax Gemini EM instrument The Instrument Settings dialog box for the Gemini EM includes choices for Bottom Read and a 325 nm additional cutoff filter total of 16 cutoff filters for the Gemini EM the other Gemini instruments have 15 The choice for Time Resolved Fluorescence has been removed from the settings for the Gemini EM SoftMax Pro User s Manual A 11 Appendix A SoftMax Pro Version Specifications t A 12 SoftMax Pro User s Manual Appendix B Glo
155. tt tte tte tte ne ten ne nae ae ae te ne ae e meme ns ne on ne aene ase tse ee ete tte ete ete ne ten tme nene ae tae tisse sse te tte te tte ete i FRRRRRRRRRERRRERRRRRRRRRRRRRRRRRRRRRRRRRRRERRRRRRRERRRRRRRRRRRERRRRRRRRRRRRRRRRRRRERRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRERRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRERRRRRRRRRRRGRARyRyR RR Ram Ram am am auam amu auum aua LLL OL CLO L LLL L LLL CLL LCL LLC L LLL LLL LL Lon es kinetic Reduction SELES MERCER CRC ER ELLE CEE Reh RCL LEC EER LE ee EE HCE LE RCL RCH RCL T CHE chE ts Ht Retin ec LER ch cL RC LEE rLE MCLE Ret ERC RCE Rech COLE RCL ECCI MCLE RcEERC HCE ER CH CCLERGEERY 30 er Limits POTEET Per CERT POTEET ra Te or T cher rege CT rca peepee notre barrie peer roa orca Y Baseline DE T Min RFU Lag Time fo C Absolute Max RFU sog End Time Zero Baseline E Points PSE Er M RR d Mee M one ote ap rd m som Sinoathing mmm TE e Baseline h Multiplier RERRREERRESERHESERRRERRRSERRERRRRERRRERRRERRRERRRSERRRERRRERRRERRRRERRRERRRRERRRERRRSERRRERRRERRRERRRERERRESERRERERE Moving Average Points Figure 10 58 Reduction Dialog Box Showing All Values Click to close the dialog box Step 5 Set the Display Parameters Click the Display button to open the Display dialog box a fee Raw Faduced hax Min Lm1 Example Options QERARRRESEEERERERESESEENERRSASERRERRERSSERERERREE x OE High s B h 0 330 uesnsnuunsneusunensuruuensu
156. until all tips and columns have been targeted SoftMax Pro does not fill beyond the limitations of the available Tips and Compound columns Once all available tips and compound columns have been assigned SoftMax PRo simply stops assigning targets This tab section will show one two or three transfers with blue pink and green color coded tips to match the setting made in the Compound Transfer tab and two pop up menus Tips Column and Compound Column The Tips Column menu will show a sequence of numbers that match the selected col umns in the Pipette Tips tab The Compound Column menu will show num bers from 1 through the number of columns of the compound plate or the number of troughs selected in the Compound Source tab A Tips or Compound row will have potentially 12 columns to represent the number of columns in a 96 well assay plate The actual number of columns and their placement further depends on the number of columns and their locations as selected in the Wells to Read tab To assign a tip to an assay plate well highlight one or more tip cells and select one of the items available in the Tips pop up menu The tip column selected in the pop up menu will be shown as a number in the selected Tip row s Choosing the Fill option at the top of the menu will fill the selected cells with the tip numbers that correspond to the same column numbers tip 3 with column 3 etc Note that Fill does not take into account the actual tips tha
157. values read by the instrument will appear in the data display of the Plate section as they are received from the instrument in real time Depending upon the read mode the number of wavelengths the plate type and the dis play options selected the data display will show one or more numbers or plots You can enlarge the display of one or more wells while the a Kinetic or Spec trum reading is in progress Double click a single well to enlarge its display to a Well Graph or hold the Shift key while selecting more than one well and then choose Graph from the Plate menu or click the graph button on the tool bar to display the data from multiple wells in one enlarged Well Graph The data in the enlarged Well Graph will update to show new data points as they are received from the instrument Other choices and settings within SoftMax Pro affect the way in which data is displayed the type of reduction specific time settings and so on These set tings are discussed in Chapter 8 Displaying and Analyzing Data Using Flu orescence or Luminescence Instruments 5 52 SoftMax Pro User s Manual Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator Ente prise uod ose x eb c pU IS Pee eI ead LS URN e edd 6 2 General InformiatioD sco o d tai e oben aera eae id 6 2 Pier prise Xd mis t6arOf oats yo mem Che ec pv eres e v ea E es 6 3 Preinstallation Checklist sc uia wk a Ge Een RU xot 6 3 Pre Installation Information
158. with seconds e New robotics commands have been added to automate opening and closing the tip and compound drawers of the FlexStation instrument e COM port switching for multiple instruments is now available e STATUS has been clarified application vs instrument busy status e A user name and password can be entered to open a password protected file FlexStation Enhancements e 384 well plates are now supported for both reading and for use as a com pound source SoftMax Pro User s Manual A 9 Appendix A SoftMax Pro Version Specifications L 3 Well Scan Mode e Blank subtractions are performed differently previously a point to point subtraction was used now all points in the well are averaged and that aver age is subtracted from the average of all sample points Export Function Updates e Notes sections are now exported with data from Plate sections e Molecular Devices copyright information added to the bottom of exported data e Exported information is formatted with tabs between items so that it can be opened and viewed using Excel and the data will parse properly into col umns Multithreading of Serial Input PC only PC serial input output is handled using a separate thread in order to enhance serial communication performance Macintosh Communications Toolbox Support e Macintosh serial communications through a USB to serial port have been enhanced SpectraMax Gemini and Gemini XS Enhancements e Ability
159. with the pre read plate blank or to override this and read the normal plate After reading the blank plate you must start the read process again for the normal plate The same dialog box will appear but the default option button choice will be Normal SoftMax Pro User s Manual Reading a Cuvette SpectraMax Plus and Plus Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Select Section Sections Plates 6 6 Pre read S Normal Figure 4 38 Select Plate Dialog Box for Pre Read Plate Blanking If you select a Plate section that already contains data and choose to Read again an alert appears asking you to confirm replacing data in the destina tion plate see Figure 4 39 If you select Replace the reading will proceed and the previous data will be replaced with new data Replace data in Plate 1 Cancel Replace Figure 4 39 Alert Regarding Replacing Data for Plate Section 384 only With the SpectraMax Plus series instruments two types of readings are possi ble using a cuvette a reference and a sample reading Reference Reading SoftMax Pro User s Manual As in a spectrophotometer taking a reference reading can be done either on air or using a cuvette containing the buffer of your sample The reading taken using using the button in the CuvetteSet section tool bar or the Refer ence command from the Control menu will be used as Ip to calculate the abs
160. you can choose Suspend Recalculation to delay calculations until a later time Displaying Data in Group Graph and Notes Sections Group Sections SoftMax Pro User s Manual The manner in which data is displayed in a Group section depends on the type of reading that was performed Endpoint Kinetic Spectrum or well scan but can be customized by choices made in the data display reduction settings and the Edit menu e Resizing hiding columns e Adding deleting columns e Showing editing column formulas e Adding editing summaries e Changing text style Default displays for Group sections are shown automatically unless you choose a different display option The particular font to be used in a Group section table as well as its size and style can be changed by first selecting one or more columns in the Group sec tion table and then choosing the Text command from the Edit menu and mak ing the desired choices from the dialog box that appears Settings from the Plate CuvetteSet Section Affect Data Display in Group Section Tables Reduction options in the Plate CuvetteSet section allow you to display the raw data received from the MDC Microplate System instrument in a reduced analyzed form Which reduction options are available depend on the read mode used and reduction settings chosen Reduced number from the Plate CuvetteSet section is displayed in the Values column of the Group sec tion tables by default Reduction
161. you to choose from the listed topics Clicking a topic opens the help screen associat Contents ed with it Index Using Help Index Shows the alphabetical listing of all items that can be viewed within About SOFTmax Pro the Help function Clicking a topic opens the help screen associated with it Using Help Allows you to choose from an indexed list of topics or to search for the type of help you re looking for About SoftMax Pro Opens the About screen which gives the serial number ROM version and the instrument type connected to the computer as well as the software version for SoftMax Pro Help Menu Macintosh About Help Brings up a dialog box explaining how to use the Apple Guide which gives detailed information about the help function on the Mac intosh About Balloon Help Show Balloons SOFT max PRO Help Formula Help Show Hide Balloons Depending upon whether or not you have balloon help turned on or off this menu presents the choice to reverse the setting SoftMax Pro Help Opens the help section devoted to SoftMax Pro Items are presented in alphabetical order Formula Help Opens the formula help section which provides information about how to use formulas including formula examples SoftMax Pro User s Manual 3 39 Chapter 3 SoftMax Pro Overview Interprocess Messaging HN This feature is available on the Windows Operating System only SoftMax Pro can
162. zi Datati uU Diatatlt 3 Datani E Data F Datatl i natat H Batai Figure 10 53 Template with First Samples Entered 10 48 SoftMax Pro User s Manual t Chapter 10 Tutorials B C SoftMax Pro User s Manual The selected wells should now show the default first sample name for the Data group Data01 The area in the top center of the Template Editor will become active showing the default values for Sample name and Dilution Fac tor In this tutorial the dilution factor for the first column of samples in the Data group should be set to 0 0 ug mL the default value of 0 is correct Click the Assign key or press the Return key on the keyboard to ensure that this con centration is assigned to the selected wells in the template You can check the concentrations you have assigned to the template by pressing and holding the CTRL Shift keys on either Macintosh or Windows This causes the display to show only group names and values assigned to specific wells Now select wells A2 through H2 Data is still showing as the default group name but a new sample descriptor is created automatically Data02 so all you need to do is assign a dilution factor of 0 01 ug mL to these wells After entering the value click the Assign box or press the Return key to assign this value to the selected wells The dialog box should now look like the illus tration below m Experimentz i Plate 1 ca Sample Data02 L
163. 0 000 0 0 Stas O00ezjcS ooog 410 000 0 000 0 0 jSta amp 0 081 ce ooog 410 000 0 000 0 0 Smallest standard value 410 000 Largest standard value 420 000 Figure 8 34 Standard2 Group Section 3 Anew Graph section is created by selecting New Graph from the Experiment menu This causes the Plots dialog box to open automatically The default name for the first plot Plot 1 is then changed by typing a new name over it to Standard 1 Clicking OK opens the Graph Options dialog box which allows you to choose the graph type modify the fonts used for the graph and so on Choices should be made as follows Standard1 for the group to be plotted the concentration of Standard1 for the X axis and values of Standard1 for the Y axis No error bars are added 6 48 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Graph Options Title Graph 1 Height Type onts ud Sample Text Eh Cluster bar Sample Text Set hil J Stack bar Legend Connect points 5 le Text Plot symbols Plate pee o Standard 4A Concentration Standard Y Yales Standard amp Errors Wo Error Y Errors Mo Error Figure 8 35 Graph Options Dialog Box with Plot Name Changed to Standard 1 4 A second plot is added to this graph by clicking the New button in the Graph Options dialog box After clicking New a dialog labeled Plot 2 is o
164. 0 341 Figure 7 47 Graph with 4 Parameter Fit 59 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 60 v Z Graphat Fit Log Logt Graphz 1 concentration y CA DMTHGUCPBME A B e D Plot 1 Grouped concentration vwa Meanvalue 0 1 1 247 11 262 0 305 Figure 7 48 Graph with Log Logit Fit Some publications show the log logit equation in another form log e bx b log c where y L4 x log A different algebraic expression of these is y EE D x C The difference between the log logit fit and the 4 parameter fit are in the way the coefficients A and D are calculated For the log logit method the standard values for the lowest and highest values of x are used The corresponding y values are assigned to A and D respectively Based on these fixed values for A and D the algorithm then computes values for B and C This technique works well if there are standard points along the upper and lower asymptotes If this is not true the log logit fit should be avoided in favor of the 4 parameter logistic In SoftMax Pro 2 x the 4 parameter logistic curve fit uses 2000 iterations If the fit does not converge in this number of iterations the program will report a fit error The curve fitting algorithm for the 4 parameter equation was based on the Levenberg Marquardt Method Discussion of this method can be found in Numerical Recipes in C The Art of Scientific Com
165. 0 43 Instrument Settings Dialog Box Showing Compound Source 10 44 Instrument Settings Dialog Box Pipette Tips Layout Full Rack Chosen 10 46 Instrument Settings Completed 0 cece ee 10 47 Group Settings Dialog Box for Data Group 0 0 cece ee eee 10 48 Template with First Samples Entered 0 0 00 c eee cee eee eee 10 48 Template showing First Samples Data Concentrations 10 49 Template Filled with Samples 00 cece eee cence teenies 10 50 xix Figure 10 56 Figure 10 57 Figure 10 58 Figure 10 59 Figure 10 60 Figure 10 61 Figure 10 62 Figure 10 63 Figure 10 64 Figure 10 65 Figure 10 66 Figure 10 67 Figure 10 68 Figure 10 69 Figure 10 70 Figure 10 71 Figure 10 72 Figure 10 73 Figure 10 74 Figure 10 75 Figure 10 76 Figure 10 77 Figure 10 78 Figure 10 79 Figure 10 80 Figure 10 81 Figure 10 82 Figure 10 83 Figure 11 1 Figure 11 2 Figure 11 3 Figure 12 1 Figure A 1 The Plate Section Showing Group Boundaries 0 eee eee eee 10 50 Reduction Diallon DOXadu saquen dar aun dq ease nea eeean regs a 10 51 Reduction Dialog Box Showing All Values lees eee eee 10 52 Display Diglot BOX 61 24 c40nessenys bere pisetesseese ea gd npa d pop iod ips 10 52 Save As Dialog Box Macintosh 0 0 0 cece cee cee eens 10 53 Save As Dialog Box Windows 0 0 c ccc eee
166. 000000000000000000000000000000000000000000000 0000000000009 Vmax units per sec w Max Points 34 M Limits So EE Sn AUT EEG MnRFU p Lag Time jV Absolute Values Max RFU ooo EndTime v C3 Plate 1 E Templ E E Cicer a os E LUE i DT Vmax units per sec p 1 2 3 4 5 6 7 8 9 10 11 12 WY yy B L L P cL L L7 A 4 Z1 Fluorescence E Pi 4 7 Bottom read T r7 re o E b u Ex Em Cutoff ci 4 k Lint 485 538 530 l7 V V Wavelength Combination Lint Lag Time 0 00 End Time 5 00 RFU Min 0 RFU Max 10000 Vmax Pts 34 34 Plate Last Read 10 27 AM 1 22 2001 Figure 8 16 Kinetic Reduction with Absolute Values Enabled Note that the limits in this example MinRFU 0 MaxRFU 10 000 do not allow all of the data to be visible To see more data yet still display absolute values enlarge the limits for MinRFU RLU and or MaxRFU RLU found in the Reduction dialog box Figure 8 17 8 24 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments RLU Limits m Reduction eduction Chinn E i Max Points 34 bo 1r Absolute values Cancel Figure 8 17 Limits in Reduction Dialog Box RFU shown If you increase the MaxRFU RLU setting to be at least as large as the largest fluorescen
167. 05 5 7 Instrument Settings Dialog Boxes for Spectrum Mode 005 5 8 Instrument Settings Dialog Boxes for Well Scan Mode 04 5 9 Instrument Settings for an Endpoint Reading 00 0 0 00000 5 10 Instrument Settings for a Kinetic Reading 0 0 00 cece ee eee 5 11 Instrument Settings for a Spectrum Reading 00 0 e eee eee 5 12 Instrument Settings for a Well Scan Reading 00 00 e ee eee 5 13 Wavelength Settings PR 5 14 Wavelength Selection for a Spectrum Reading 0 e eee eee eee 5 15 Cutott Seines WING OW ine aree da tr teste segues Words troia q fed deed d Rudd 5 15 Numberof Readings Seng suce schien mae meto per RACE POP or dor ree a er 5 16 PMT Sensitivity Setting SpectraMax Gemini fluorescence readers 5 17 JAUTOPDDCDOTLUBPS DOX essy een rE ERNIE EESE border top e E e Rudi an ee bate 5 17 Isai uo ct ui EET erm 5 18 SoftMax Pro User s Manual Figure 5 16 Figure 5 17 Figure 5 18 Figure 5 19 Figure 5 20 Figure 5 21 Figure 5 22 Figure 5 23 Figure 5 24 Figure 5 25 Figure 5 26 Figure 5 27 Figure 5 28 Figure 5 29 Figure 5 30 Figure 5 31 Figure 5 32 Figure 5 33 Figure 5 34 Figure 5 35 Figure 5 36 Figure 5 37 Figure 5 38 Figure 5 39 Figure 5 40 Figure 5 41 Figure 5 42 Figure 5 43 Figure 5 44 Figure 5 45 Figure 5 46 Figure 5 47 Figure 5 48 Figure 5 49 Figure 5 50 Figure
168. 1 cm pathlength PathCheck is covered by U S Patent Number 5 959 738 384 Photomultiplier Tube PMT A vacuum tube that detects light especially from dim sources through the use of photoemission and successive instances of secondary emission to produce enough electrons to generate a useful current Pipette Tips FlexStation only The FlexStation instrument requires the use of pipette tips which are kept in a separate instrument drawer Molecular Devices suggests that you load a com plete set of pipette tips each time you make a run to ensure that sufficient tips are always available Please see the instrument manual for the FlexStation regarding the types of tips that can be used with this instrument and how they should be loaded Plate Section The Plate section is used to display data to specify how microplates will be read and to define how the data received from the instrument should be reduced The data display in this section is a matrix that corresponds to the well format of the microplate in use Plate Type A choice in the Instrument Settings dialog that determines the type of micro plate being read The specification of the plate type depends on the well for mat and manufacturer of the microplate Point Using the Mouse You point to an object on the screen by moving the mouse so that the pointer is positioned on that object When the pointer is an arrow the tip of the arrow must be on the object Point to Point
169. 15 7 4 data display 4 45 5 36 5 52 7 4 7 5 8 5 10 22 10 54 instrument settings summary 4 5 5 5 menu commands buttons 3 16 reduction options 7 4 8 4 Plate Settings command 3 34 Plots 7 47 7 54 8 45 8 52 command 7 47 8 45 PMT sensitivity 5 43 Point to Point curve fit 7 61 8 59 B 11 Port B 11 Preferences 2 16 command 3 29 9 9 Pre Installation Enterprise Administrator 6 5 Pre read plate blanks 4 19 4 33 4 34 5 30 5 31 B 2 B 3 copy paste data 4 15 Print command 3 27 9 14 Print Section command 3 27 Printing 9 11 Autoprint 9 11 options 9 11 problems 11 14 report 9 11 9 12 10 34 10 67 including excluding sections 9 12 section 9 11 order 9 12 setup 9 11 template 9 11 well graph 7 21 8 17 9 11 PrintShop Conflict with SOFTmax PRO 11 15 privileges 6 14 Protocol file 2 9 9 4 9 5 default 9 3 B 5 locating 9 5 saving 10 21 10 53 Q Quadratic curve fit 7 58 8 56 B 12 Quit command 3 27 R Radio button option button B 10 RAM Random Access Memory B 12 Ranged display 7 15 7 16 8 11 8 12 Read 4 40 5 34 5 51 10 22 10 54 button 3 8 command 3 31 4 40 5 34 5 51 cuvette 4 39 4 41 5 51 entire plate 4 18 5 18 microplate 4 39 4 40 5 34 5 51 mode 4 9 5 10 5 40 Endpoint 1 3 4 9 5 10 FLEX 5 40 Kinetic 1 3 4 10 5 11 Spectrum 1 3 4 11 5 12 Well Scan 1 4 partial plate Strips 4 18 5 18 5 43 priority 4 44 5 35 strips 4 18 5 18 5 43 wells to read 5 43 Read t
170. 160 eit nr teos WaedREd PPP TAPER 2 9 Enter Registration Information sseeeeeeeeeeeeeeees 2 13 Communicating with the MDC Microplate System Instrument 2 15 v dg i P E E T H 2 16 Seal Ol cnc e2dos odo esate ie at ene qued t a drip dei 2 16 Export TOA ind Eua ese seat aes hae es eee ene ed sears pat 2217 CDI P M 2 18 Ii CD A E E A E 2 19 AO AYE 54 bot Gobo E E N 2 20 sol REPETERTE TE EEEE E T 2 22 Aayo FOMCK P atc E Goi Gh as a Senet 4 oe 95 2 23 Chapter 3 SoftMax Pro Overview eee 3 1 lovers ibronlo SEPT 3 3 The SoftMax Pro Window 00 ecc cece eee cence eeees 3 3 june dr oM m 3 6 wirds METTI a S E E 3 7 Experiments and Secti nis sss seserinis is deed na Ghee eA ad 3 8 EXDEEHBGDEIS 444544 cohetes PIPER ODE RP A E 3 8 ese O p anes 3 9 INOtGS DOCHOPL deu eos ond Poe E UR Red dci ubt tue ciu d 3 13 Plate Section MET 3 15 CV ete et DeCHON 212 quoe ega 9n OR a a e eee S 3 17 CITOUD De CHOON o edenda E E E ET 3 19 funes P e a ei aR 3 21 Meramente Sosa a a e ddr pe AR pied 3 23 Menus and Commands 0 cece eee eee eens 3 23 PODS MCUs sano 3 26 About SoftMax Pro 2 2 eee eee eee 3 26 File Meni T 3 26 EUEN Deere idee E 3 28 NCW E PR E EEE ET 3 30 EXDEFHBICBDCDTOB Uses idet 1b 2 89 Gadde ena 3 30 Control Menu a ood cia ico torte tpa aep pene one erdpd in ida f perd 3 31 PSSA So MED Pr 3 32
171. 24 231 ndi Rat ond dae d p ER RS es 5 40 oeltings TOF PLEX MO si rort a EPI uat Ime teta 5 41 Template Edito sersan as sass geal SEP diy aes eae aid Bete 5 51 Readine a MICFODISI soda E PER rp Ride ki emer RES 5 51 General Information about a Reading 5 52 Chapter 5 Reading Using Fluorescence or Luminescence Instruments t 0 2 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Introduction 1 2 3 4 5 6 7 8 9 10 11 Reading a microplate using SoftMax Pro is a three part process e Prepare SoftMax Pro to collect data e Prepare the microplate s to be read and e Read the microplate s Before reading a microplate you must create the Plate section s and define the instrument settings for the reading Instrument Settings MUST be defined prior to collecting data and may NOT be changed after data is collected In addition it is recommended that you define a template and set the reduction and display parameters prior to reading the microplate since these parame ters determine how data is displayed and analyzed but these last parameters may be set up and or modified after data collection A typical process for preparing collecting and analyzing data is as follows Start SoftMax Pro Create Plate section s as needed Define instrument settings use buttons in the Plate section tool bar or the Control menu Define the templa
172. 4 MFC C Interface to SoftMax Pro Remote Commands that Rebar ValueSss s asco sandpx erede Gre E REUS E RCD 12 15 Chapter 12 SoftMax Pro Remote Command Language t 12 2 SoftMax Pro User s Manual Chapter 12 SoftMax Pro Remote Command Language Introduction SoftMax Pro can be controlled by other Windows applications by means of a simple ASCII string based messaging system Using the Win32 SendMes sage function SoftMax Pro remote command strings such as ReturnStatus Read or SaveAs myFile may be sent as a Windows Message to SoftMax Pro for remote process execution Additionally SoftMax Pro version 4 0 1 or later supports queued processing of remote commands This feature allows the client process to send a block of remote commands and then pause for completion as opposed to testing for command completion between each command The user is never blocked or held up while sending remote commands An additional benefit of this option is that when the client processes the request status the ReturnStatus command is processed immediately even if SoftMax Pro is busy carrying out other tasks A remote command Using SendMessage U function either WM COPYDATA or ser enabled WM SETTEXT command is sent AASEN Client Process Text data returned asynchronousl Next remote result of WM COPYDATA being used command to execute v SOFTmax PRO Process result of WM SETTEXT being used Text data returned syn
173. 4 PM 2 5 2001 1e3 Je Se 5e3 EE Teg mE gez Bd 1e4 B 124 E 1e4 Figure 8 8 Endpoint Data Reduced Displayed as Gray Scale Graphing Wells You can enlarge the display of the wells shown with Kinetic Spectrum Well Scan or FLEX modes to get a close up view of the data This is done by either double clicking the particular well in the data display or by selecting the well and then clicking the Well Graph button in the Plate section tool bar Kinetic Spectrum and FLEX Well Graphs Well Graphs plot the data for the individual wells in a microplate and show the well ID and reduction information including the goodness of fit R 2 value if appropriate The default display for the X axis shows time for Kinetic and FLEX plots and wavelength for Spectrum plots Positioning the cursor within a Well Graph shows X and Y values for the cur sor location allowing you to determine more precisely the location of data points Well Scan Well Graphs Well Graphs in Well Scan mode default to displaying raw data in a grayscale plot The manner in which the data is displayed in a Well Graph for a Well Scan depends on the reduction options that have been chosen the display options raw or reduced for example the number of points read and whether the Well Graph is scaled to the data or to the limits of the entire plate 8 14 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence I
174. 5 Wavelength Selection for a Spectrum Reading 0 ee eee eee eee 4 13 Speed Read Section of the Instrument Settings Dialog Box 4 13 Instrument Settings Showing the PathCheck Option uuesue 4 14 Automix Settings Box SpectraMax and VersaMax Instruments 4 17 Automix Settings Box for Vmax UVmax and ThermoMax 4 17 Partial Plate Read Strips Dialog Box with Eight Strips Selected 4 18 Blanking Section of the Instrument Settings Dialog Box 4 19 Template Editor Dialog BOX iren ne mt red she eee Sool ad does 4 21 The Appearance of the Template Editor Tool Bar under Different rers 4 23 The Group Drop Down List eeeeeeeeeeeeee e 4 24 Group Settings Dialog Box Using Default Settings 4 24 Group Settings Dialog Box for the Group Named Patient 1 4 27 Groups Settings Dialog Box Showing Original Column Format 4 27 Modifying Sample Names and Descriptor Values in the Template Editor 4 28 The Sample Drop DowtLbiSb osc ntssntbodatescddatada Tu reped e intus 4 29 Sample Drop Down List Sample Names Defined and Wells Selected 4 29 Assign Area of the Template Editor Tool Bar 0 00 ee eee eee ee 4 29 Template with Wells Selected for a Series 00 c eee ee eee eee 4 30 Senes TIBIOS BOX dkea seep anes SI
175. 5 command 3 33 3 35 connect points 7 65 8 62 copying 9 25 curve fit 7 53 7 54 8 51 8 52 error bars 7 53 8 51 formulas 7 47 8 45 menu 3 37 options 7 65 8 62 axes 7 67 8 64 bar 7 66 8 63 button 7 19 8 16 defaults 7 65 8 62 scatter 7 65 8 62 standard curve 10 31 summary 7 47 8 45 Graph section 3 10 3 21 7 47 7 54 8 45 8 52 curve fit 7 53 7 54 8 51 8 52 menu commands buttons 3 22 plots 7 54 8 52 Graph Type command 3 37 7 65 8 62 Graphing wells 7 18 8 14 Gray Scale display 7 15 7 17 8 11 8 14 Group 3 10 3 19 10 25 10 57 blank plate blank 10 12 boundaries 4 21 4 22 5 20 5 21 columns 10 25 10 57 adding 7 45 8 42 copy paste 9 27 formulas 7 43 7 45 8 40 8 43 customizing 3 10 menu 3 38 sample descriptor 4 31 5 29 sample name 4 31 5 28 series 4 31 5 29 Template Editor 4 20 5 19 text format 9 14 units 10 12 Group section 7 41 8 38 menu commands buttons 3 20 Group Settings command 3 38 Group Settings dialog box assign area 4 29 5 27 sample area 4 28 5 26 sample descriptor 4 25 5 23 Group associated blanks 4 33 4 34 5 30 5 31 B 2 B 3 Groups Page 6 11 l 4 H Help 1 9 1 10 formula 1 9 hypertext links 1 12 search function Windows 1 11 Hide command 3 38 7 42 8 39 Hide Formulas command 7 43 8 40 Hide Replicates command 3 38 Highlight B 7 I Icon instrument 2 15 Idle Time out 6 10 iMac 2 4 Import 9 21 Criterion 9 21 data 9 21 FLIPR 9 21 Impo
176. 6 8 448 HeanUalue 0 105 0 129 0 157 0 192 0 234 0 28 0 349 0 427 The first line of an exported ASCII data file includes one field indicating the number of data sets in the file This single field includes the text BLOCKS followed by a number that equals the number of data sets The second line of a file for a Plate or CuvetteSet section includes 19 fields that provide all of the information needed to determine the type of test that was run to measure the first data set The following table describes each of these fields 9 19 Chapter 9 File Management and Printing l B Table 9 2 ASCII Data File Fields and Descriptors Sectionkind Either Plate or Cuvette Plate or Cuvette PlateFormat or set in Preferences Endpoint Kinetic Spectrum Well Scan or FLEX Data mode For absorbance plates Absorbance or Transmittance For others Fluorescence Luminescence or Time Resolved Fluorescence Data type Raw or Reduced 8 Pre read included TRUE or FALSE always FALSE for Cuvettes Kinetic points Reduced plates and Endpoint plates Kinetic Spectrum plates number of Kinetic or FLEX read Kinetic FLEX read time in seconds or Well Scan read pattern horizon time Well Scan read tal vertical X or fill otherwise blank field pattern 11 Kinetic or FLEX Kinetic or FLEX read interval in seconds or Well Scan density otherwise interval Well Scan blank field density S
177. 605 0 669 0 739 0 817 0 903 0 297 Wavelength Combination Lr1 Data Mode Absorbance t 01 L1to1 2 Figure 7 10 Endpoint Data Displayed as Threshold with Reduced Number Ranged When the Ranged display is chosen raw data that falls between the high and low limits is assigned proportionally to integer values from 0 through 9 Val ues above the high limit are displayed as a plus and values below the low limit are displayed as a minus Figure 7 11 shows reduced Kinetic data dis played in Ranged format v Gy Piate i 4 Plate 1 Kinetic Time 5 Tu Inter yal 0 a Lmi 450 Automix OTF Calibrate Once OG Max 1 Wavelength Combination ILm1 Ymax Pts 34 34 Data Mode Absorbance I nm m c oOo uU F Plate Last Read 9 16 AM 7 16 23 3 43 O zzz 1 404 zZ x 485 3 x 565 4 lt 646 3 TZT 6 808 T 888 5 lt 969 9 lt 163 gt le Figure 7 11 Kinetic Data Reduced Displayed as Ranged 7 16 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments SoftMax Pro User s Manual Gray Scale The Gray Scale option presents the raw data in eight shades of gray changing from light for values less than or equal to the low limit to dark for values greater than or equal to the high limit Figure 7 12 shows reduced Endpoint data displayed as Gray Scale v Ej raten Eria E 74 Plate 1 Endpoint amp utamix OFF Calib
178. 7 5 00M columns 100b columns 10uh column10 10040 The calculated EC 50 fram the Data section EC50 5z Ea Disa L 4 b Graph Fit zs tz o Figure 10 43 Default Protocol with Notes Section Open SoftMax Pro User s Manual 10 37 Chapter 10 Tutorials The experiment should have a Notes section Notes 1 followed by a Plate section Plate 1 one group section Data and a Graph section Graph 1 that will be used in this tutorial For this tutorial you will recre ate the settings in this file To start with a clean slate you must first delete some of the existing sections Scroll down so that you can see the rest of the sections in the experiment Hold down the Shift key and click on the Plate 1 Controls and Data sections From the Edit menu choose Delete Selection A message box will appear asking you to confirm that you wish to delete these sections Choose Delete and the sections will be removed from the experiment Now you must create a new Plate section Choose New Plate from the Experi ment menu A new section named Plate 1 will be created Step 2 Define the Instrument Settings 10 38 Scroll down and click the Plate 1 section to make it active its tool bar will become gray Then click the Setup button in the tool bar to open the Instrument Settings dialog box Endpoint is selected by default This tuto rial uses FLEX mode so you must c
179. 7 50 Graph with Cubic Spline Fit 7 62 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments SoftMax Pro User s Manual Exponential The exponential function used to generate this curve fit is y A B 1 exp x C EARN o MEME Graphz B concentration y A B 1 exp xic yy A B c Ree Fiol iGroupz1 concentration vs Mean value 0 128 0175 28 984 0 363 Figure 7 51 Graph with Exponential Fit SoftMax Pro does not plot samples with descriptor values of zero 0 on a log axis Curve fits affected by this are the semi log log log log logit and 4 parameter curve fits 63 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 1 B Minimum Number of Standards The number of standards used in a standard curve depends on the particular curve fit selected and on the judgment of the operator The table below gives the minimum number of standards required mathematically by each of the curve fitting algorithms Table 7 4 Minimum Number of Standards Required for Curve Fits ter Qmm 3 site KENN mm 0 0 RO AINI NINJ N de 0 00 emer 000 4 Reemi 0000003 0 EL RN mem o miewl ny For optimal results you should always exceed the minimum number of stan dards required for any given fit There are other requirements on the standards for the log logit and 4 parame ter logistic curves The fits for the 4 parameter lo
180. 9 GEP GMIP SUPPO GS Lete pacseriuino dup Bett oam bie a ree adobe A 9 CHECTIODAEMOS ausu hays bt yet ties ors eed aa eee datos A 9 FEIER TIIS otc xai e dune merece sang aUe os a ghee A 9 RODOS aceea Ge contin dam Sidi teh eta A 9 FlexStation Enhancements 0 20 e eee eee eens A 9 Well Scan Modesa 4r obey ot uq re hay vedi o scita A 10 Export Futicion Updates sirgete ting ao e o in Ro Pas ase A 10 Multithreading of Serial Input PC only A 10 Macintosh Communications Toolbox Support A 10 Appendix A SoftMax Pro Version Specifications L 3 SpectraMax Gemini and Gemini XS Enhancements A 10 Enhanced 4 Parameter Fit slllsleeee leue A 10 Documentation amp Software Enhancements A 10 Specifica ONS uniebes scire ir ediars ung Ies er ard aS Rea Ead dad A 10 o5oftMax Fro 4 1 SpeCincaiOn sura oe rd mre ERE P er A 11 vjerselr ec quio a PORT A 11 A 2 SoftMax Pro User s Manual Appendix A SoftMax Pro Version Specifications Introduction This Appendix lists enhancements and bug fixes made to SoftMax Pro in pre vious releases SoftMax Pro 3 1 Update Specification SpectraMax Plus one support The SpectraMax Plus was added to the Reader list in the Preferences dia log The Instrument Settings dialog for this new instrument is very similar to that for the SpectraMax Plus with the exception that a Plate Type options was included The Plate Type se
181. 959 Once Costar 3961 Assay Plate Type Costar ntm Greiner Vbtm 96 Well Standard Polyfiltronics 7701 5400 Wells To Read Read entire plate Compound Source Beckman 140504 v Compound Transfer Compound Transfer T1 100 1 30 10 Triturate Triturate Assay Plate Fluid T1 Not used Initial Volume p Jul Pipette Tips Layout wj Full rack M Transfer 1 Compound amp Tip Columns rines T 14 22 2 3 3 5 el Compound Sources i ri Assay Plate eee 4 4 4 5 5 5 5 5 6 s l ipette Height Tine Point i7 secs 7 7 7 8 8 8 9 9 8 Po voum Rl ii volume Jui Volume ul DONE 10 10 10 11 14411 Ae ii cyes Rate i ycles my 12 12 2 IB icht Ju i BS a Off Compound amp Tip Columns Pipette Tips Layout Tips Column Compound Column Yy 1 2 3 4 5 6 Wy 8 8 10 11 12 J RRR eae eRe ERE ECT ERA AutoRead Turn autoread on Delay n secs Figure 5 36 Instrument Settings Dialog Boxes for FLEX Mode SoftMax Pro User s Manual 5 99 Chapter 5 Reading Using Fluorescence or Luminescence Instruments k Read Mode To select FLEX read mode click the appropriate button along the top of the Instrument Settings dialog box m Instrument Settings Ol l e ei Fa Endpoint Kinetic Spectrum Well Scan FLEX Read Mode Buttons Options Wavelengths al Wavelengths Ex Em Auto Cutaff On
182. ALLY and you will not be able to automatically re open the data file in SoftMax Pro If you choose to save to this format you will need to save your data manually using the Save or Save As commands in the File menu if you also want to save a SoftMax Pro file If you attempt to quit SoftMax Pro before saving a file containing data that has not yet been saved a message box will appear asking if you want to save the file You can assign any name to a file you save manually Nesey If your protocol contains more than one experiment section when you Autosave to SoftMax Pro file format each experiment will be saved to a separate data file NOH Enterprise Administrator can update and modify the autosave parameters of Softmax Pro Enterprise Edition When Autosave is enabled the box below the Autosave option becomes active allowing you to choose the folder within which data files will be saved and the type of file to be saved You may choose to autosave as a SoftMax Pro file default or as a text file and when saving as a text file you may choose to append to the same file The default folder for saving files is the SoftMax Pro 4 3 folder To save data files in a different folder click the Set button A dialog box will open allow ing you to choose a new location for saved files Select Directory X Director c saftmax pro Select Button Cancel Drives E c CORME Figure 2 14 Autosave Location Dialog
183. All columns that were hidden will again be visible There is no way to show only one hidden column Enlarging Column Size You can enlarge the width of columns to any size Increasing the size of col umns may make the width of the Group section increase beyond the page width boundary If this occurs the columns will disappear off the edge of the screen but are really still there To bring these columns back into view reduce the width of the columns that are still visible until the others reappear Standards g ml Sample Concentratian wellis Values Meanvalue Std Deje Divider being dragged to the left Figure 7 29 Dragging a Column Divider Line 7 42 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments If the width of a section extends beyond the page width boundary the portion that is not visible will not be printed To size two or more columns to the same width highlight those columns and then drag one of the dividing lines to the desired width The width of all high lighted columns will be sized the same as the column to the left of the divid ing line you are moving Columns can also be resized to the minimum amount of space required by choosing Autosize from the Group menu By highlighting the entire table or individual columns and then choosing Autosize the amount of space each column requires will be reduced to the minimum size required by the largest pi
184. Analyzing Data Using Absorbance Instruments The CuvetteSet section offers three display choices e cuvette per row e 3 cuvettes per row default e Microplate format 8 x 12 array v EB resar Z CuvetteSet 1 Endpoint Lm1 450 Data Mo Data Eef Wo Reference Wavelength Combination Lrm1 Data Mode Absorbance v ED cuvetteset Enz CuvetteSet 1 Endpoint Lmi1 450 Data Mo Data Data Mo Data Data No Data Ref Ma Reference Fef Wo Reference Eef Mo Reference Wavelength Combination ILm1 Data Mode Absorbance 7 E cuvettesett k F4 CuvetteSet 1 CL LOTO LI LI IG IG qe9 T MEE I n om Y O U E Wavelength Combination Lrni Data Mode Absorbance Figure 7 5 CuvetteSet Display Options From top to bottom one cuvette per row three cuvettes per row and microplate format The height of the cuvettes in the 1 and 3 cuvette per row displays may be changed by highlighting any cuvette in the CuvetteSet section placing the mouse on the bottom of the cuvette the pointer will turn into a cross hair cur sor with vertical arrows and dragging down NOINA Resizing in this way will affect the display of all cuvettes in the CuvetteSet section SoftMax Pro User s Manual 7 11 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Wavelength Combination Lm1 Data Mode Absorbance A Wavelength Combination Lm1 Data Mode Absorbance B Wavelength Combin
185. Assay Example 00005 10 5 Define the ProtOCOl stre src oca Pn Roe en 10 6 Start SoftMax Pro Create a New File 0 0 0 0 eee eee 10 6 Define the Instrument Settings 000 ee 10 9 Define the Template 0 cece e ee eee ee 10 12 Set the Reduction Parameters 0 eee eee 10 19 Set the Display Parameters 0 002 cece ee eee 10 20 Read the late eacus omes ed a Uaceg RS RSS dn ores d 10 22 Data Analysis Group Sections 00 00 00 nne 10 25 Data Analysis Standard Curve 00008 10 31 Panta ISeDOEU ssri amii dco dou ede qr dur 4 iS RUE ULU ana anne 10 34 Tutorial 2 EC 50 Assay Tutorial FLEX mode only 10 35 Hard and 5oft Paramelers aue rmt arte E ps 10 35 Description of the Assay Example susesss 10 36 Define the Protocol 0 0 cece eee eee eee ees 10 37 Start SoftMax Pro Create a New File ees sees 10 37 Define the Instrument Settings lesse eee 10 38 Define the Template 0 e eee eee eee 10 48 Set the Reduction Parameters 00 ee eee eee 10 51 Set the Display Parameters lle 000 eee eee eee 10 52 Read the Plates osse pas ote diga ch pd dart quod qd Od d oid na 10 54 Data Analysis Group Sections 0 00008 10 57 Data Analysis Graph 00 cee cece eee ee 10 64 Data Analysis Summary Form
186. Autocalibrate in the second field A description of AutoCalibrate should now be displayed Instrument settings are now complete Click to accept the settings and close the dialog box SoftMax Pro User s Manual 10 11 Chapter 10 Tutorials 10 12 Step 3 Define the Template Group Pull down Menu A Click the Template button in the Plate section tool bar This will open the B SD ae Ner Template Editor showing all wells empty Drag the mouse or shift click to select the first two wells A1 and A2 Click the Group pull down menu on the Template Editor to see the group selections Choose Blank The two selected wells in the template will now be defined as the plate blank m Expernment 1 Plate T peeves Cancel OF Figure 10 4 Template with Plate Blank Wells Assigned Now select the two wells B1 and B2 Select Standards from the Group menu The selected wells will now show the default first sample name for the Standards group St01 The area in the top center of the Template Editor will become active showing the default values for concentration and units my Expenment 1 Plateit smweber e Concentration oC ER Te tt Series wr Assign Cancel OF Figure 10 5 Template with First Standards Entered SoftMax Pro User s Manual Chapter 10 Tutorials i In this tutorial the concentration for the first Standard should be set to 0 ug ml I
187. Blanks One Column of Standards and Experimentz1 Platez 1 sapie uns Te Dilution Factor io ugzrat the Rest Unknowns Run with Replicates and with Serial Dilution Standards Unknowns e Unknowns Unknowns aa Unknown Unknowns 8 Unknown 9 Unknown 10 Unknowns d Unknowns Blank Standards Unknowns 16 Unknown Unknown 18 Unknowns EE Unknowns Unknown Unknowns Unknowns Unknown Standards Unknown Unknown Unknowns Unknowns Unknown Unknown az Unknowns Unknowns Unknown J6 Standards Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Empty Standards Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Standards Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution Unknowns Dilution EL Stali Und Lind Und Unio Unia Unio Unis Unis Unis Stali Lind Und Und Ini 1 Uni 1 Un1 1 Uni Uni Uni 3 5 0 no ho ee o oh Concentration Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilutio
188. Commands In SoftMax Pro version 4 0 1 or later the user may choose between send ing remote commands using windows message 1 WM_SETTEXT or 2 WM_COPYDATA 12 4 1 The WM_SETTEXT approach Call theWin32 RegisterWindowMessage function to register a unique message tag SoftMax Pro uses this tag information when the command is received to distinguish between a Windows generated message and an external com mand The tag should be a non zero value UINT tag RegisterWindowMessage SOFTMaxProMsg All messages must be sent to the SoftMax Pro main window Note that if SoftMax Pro is not currently running this will return a null value requiring your program to discontinue until SoftMax Pro is restarted HWND hwnd FindWindow SOFTMaxProMainWnd SoftMax Pro A complete list of remote commands can be found in SoftMax Pro Com mands on page 12 9 Do not forget that the tag value from RegisterWindow Message above is sent with the command string See Microsoft documentation regarding the SendMessage function for more information SendMessage hwnd WM SETTEXT tag LPARAM remoteCommandString For example to send SoftMax Pro a Read command SendMessage hwnd WM SETTEXT tag LPARAM Read If return data is expected such as with remote commands Return Statue Keturn Timing and ReturnData the data is returned to the clipboard The advantage of this approach is that it may be universally applied to Visual
189. Concentration h a egi Sample Descriptor Figure 5 23 Modifying Sample Names and Descriptor Values in the Template Editor A ONORA When selecting wells to change sample names or descrip tors be sure all wells are replicates of the same sample and group or that you want them to become replicates of the same sample and group SoftMax Pro does not prohibit selecting individual wells from different samples and or groups for example and changing their descriptions and they will change to become the same Sample Drop Down List The sample drop down list consists of two areas divided by a horizontal gray line Before other sample names have been defined a single pre defined name BL appears below the gray line the sample name BL is the name reserved by SoftMax Pro for group associated blanks The item above the gray line is the sample ready to be or currently being assigned After being assigned it will be shown below BL below the gray line Ende Stau Concentration O ma j Figure 5 24 The Sample Drop Down List When this list first appears the only selection shown below the dividing line is BL the active sample name that is ready to be assigned appears above the line When samples are assigned to a group the default name is the first 3 letters of the group name followed by a number For a group called Standard for example the default sample names would be Sta01 Sta02 Sta03 and so on
190. Display L ZZ ia potential of SOFT max PRO Plate 1 A 1 2 3 4 5 6 ti 8 3 10 11 12 eriment 1 Endpoint Quantitative Endpoint assay with Standards and Unknowns with and without interpolated from a standard curve Fluorescence Bottom read If You Are Familiar with SOF Tmax PRO Customize This Default Proto 1 Delete any sections you do not want for example select the Cuvetteset l a 4 Wake any changes you wish to the Instrument Settings by pressing Setup instrument connected Ex Em Cutoff Lm1 470 525 495 Automix Off Calibrate Once PMT Auto c OO mom DUO um J 3 Click the Template button and assign any default or new groups as necess there 4 Select the desired Reduction and Display Raw vs Reduced parameters ReadsAMell 6 Wavelength Combination ILm1 Plate Last Read 5 Save thos default protocol with the name Default Protocol in the same folde No Data you will be asked if you wish to replace the existing default protocol choose now launch each time you open SOFTmax PRO A copy of the original default Tutorial folder if needed gt IT standards HeH Z4 F a gt Ff contrats uau 4 fi a lt C Experiment gt ETE unknowns ug Z4 fi a v Erase igiene ree E fene Uo Fit E Platez1 1 2 3 4 5 B 7 8 1 gt cC Automix Ott o Calibrate
191. Double click a single well to enlarge its display to a Well Graph or hold the Shift key while selecting more than one well and then choose Graph from the Plate menu or click the graph button on the tool bar to display the data from multiple wells in one enlarged Well Graph The data in the enlarged Well Graph will update to show new data points as they are received from the instrument Other choices and settings within SoftMax Pro affect the way in which data is displayed the type of reduction specific time settings and so on These set tings are discussed in Chapter 8 Displaying and Analyzing Data Using Flu orescence or Luminescence Instruments Reading Microplates Using the FlexStation Instrument in FLEX Mode 5 36 Reading a microplate using SoftMax Pro using the FlexStation instrument in FLEX mode is a three part process e Prepare SoftMax Pro to collect data e Prepare and load the microplate s to be read along with any other solu tions or pipette tips needed and e Read the microplate s Before reading a microplate you must create the Plate section s and define the instrument settings for the reading Instrument Settings must be defined prior to collecting data and may NOT be changed after data is collected In addition it is recommended that you define a template and set the reduction and display parameters prior to reading the microplate since these parame ters determine how data is displayed and analyzed
192. EH Windows Compatible Computer The type of cable required to connect your computer to a MDC Microplate System instrument will depend upon the type of MDC Microplate System instrument and the type of serial port s available on your computer SoftMax Pro supports up to nine connections serial ports SpectraMax VersaMax and FlexStation instruments have 8 pin DIN serial ports Emax Vmax UVmax and ThermoMax instruments have 25 pin serial ports The cable should be a straight through serial cable The serial connector for your com puter may be a 9 pin or 25 pin connector or you can use an adapter that con verts from 9 pins to 25 pins see Figure 2 3 2 4 SoftMax Pro User s Manual Chapter 2 Installation 9 pin to 25 pin adapter p connector P LL i g 9 pin connector mm mum 25 pin connectors Figure 2 3 Serial Cables for PC Top left 8 Pin DIN to 9 Pin Serial Cable top right 9 Pin to 25 Pin Adapter bottom left 25 Pin DIN to 9 Pin Serial Cable bottom right 25 Pin to 25 Pin Serial Cable SoftMax Pro User s Manual 2 5 Chapter 2 Installation l k Computer System Requirements The following computer hardware and operating system software specifica tions are recommended to ensure proper DE SISHOR of SoftMax Pro The mini mum configuration required is also given Macintosh Computer Recommended System Configuration e Apple Power Macintosh G4 e MacOS 9 1 or greater OS X in Cl
193. ENTE RIDE E edo pd aa a gu Apu d p ore eben 4 31 Fill Options and ReplicateSettings leeren 4 32 Graphic Representation of Plate Blank Subtraction 0 0005 4 34 Template Editor Showing Plate Blank and Group Associated Blank Wells 4 35 Copy Template and Paste Template Commands in the Plate and CUVENESSENIEBHISA doe 92 399 20 25 IcRO qud Od dtu quo died faded ied meds 4 36 Export and Import Template Commands in the Plate and Cavet oc NODIS oos ed edo pain E de d dod qud Venda an RU gene ans 4 37 Template Example Showing Two Blanks One Column of Standards and the Rest Unknowns Run with Replicates and with Serial Dilution 4 38 Export of the Template Example Shown in the Previous Figure 4 38 Column Data in the Exported Template Information sses esses 4 39 Select Secon Jae DON saored ms s vut sere ises chasse deed PER pd ra yas 4 40 Select Plate Dialog Box for Pre Read Plate Blanking 0 4 4 Alert Regarding Replacing Data for Plate Section 00006 4 4 Alert Regarding Replacing Data for CuvetteSet Section 4 43 Status Bar during a Kinetic Reading SpectraMax 0 0002 a ee 4 43 Calibration Dialog Box SpectraMax Plus or Plus38 and 190 340PC 4 44 Instrument Settings Dialog Boxes for Endpoint Mode 04 5 6 Instrument Settings Dialog Boxes for Kinetic Mode 0
194. Endpoint is selected by default as the read mode no change is required Clicking the setting name bold heading on the left side of the Instrument Settings dialog box shows the choices available for that setting in the right pane allowing you to make changes The table below shows the settings that should be chosen for this tutorial example for both absorbance and fluores cence instruments Following the table are instructions regarding how to choose those specific settings Table 10 1 Instrument Settings for Absorbance and Fluorescence Instruments For Absorbance Instruments For Fluorescence Instruments m Instrument Settings E x v Endpoint Kinetic Spectrum Options Automix amp Blanking i Before Off Lint kso v Pre Read Plate Off i AutoCalibrate ccm eR m c Me a j On Strips Read entire plate AutoRead Off Cancel Wavelengths 1 450 nm default Pathcheck Off default this setting only avail able with certain instruments Automix and Blanking Automix Before Off default Pre Read Plate Off default Strips Read entire plate default AutoRead Off default SoftMax Pro User s Manual mi Instrument Settings EB x 9 2 Endpoint Options E etx y z ILIA AN Fluorescence p Number of Wavelengths 1 w Ex Em Auto Cutoff On i 485 538 530 i Excitation Emission M Auto Cuttoff i Sensitivity Lm Bes vw Ese v 530 wy Read
195. Enterprise Administrator software allows an administrator to assign user levels and permissions regarding the use of SoftMax Pro Enterprise Edition e A full audit trail showing all user activities is included as part of the Enter prise Edition of SoftMax Pro when coupled with the Enterprise Administra tor software Password Protection Password protection continues to be available with all but the Enterprise Edi tion of SoftMax Pro While the Enterprise Edition will respect password pro tected files generated from other versions the option to password protect a file is not available in Enterprise Edition Shielding Shielding is no longer offered Shielded data files will revert to password pro tected files with the password scrambled so that password protection cannot be removed Shielded protocols will simply have the shielding removed Electronic Statements Electronic statements E Statements provide a way of signing SoftMax Pro files electronically with the Enterprise Edition only A new menu item under Experiments allows the user to add an Electronic Statements section to a doc ument Electronic statements allow those with permission to do so to sign a statement stating that a document has been approved or reviewed for exam ple Once a statement has been signed the document may not be modified Statements and electronic signatures are saved with the document State ments are NOT saved with protocols File extensions f
196. Example Abs 10 10 Abs 10 10 Kinetic and Spectrum ccessors Acos number Math Functions ES Returns the arccosine of a number ar list af numbers Math Operators Example Acos 0 25 1 82346 Other PathCheck accessors AntiLog number Plate Data Accessars Returns the ntiLag base eJ of a number or list of numbers This Plate Information is the same as e number Access rs Example AntiLog 13 22 718 Plate Reduction ccessors Referencing objects by AntiLog1O0 number name Returns the AntiLog t base 10 of a number or list of numbers Statistical Functions This is the same as 1 number Text Functions Example AntiLag 13210 Figure 1 2 Formula Help Window 1 9 Chapter 1 Welcome to SoftMax Pro 9 9 H Windows On Line Help You can access on line help within SoftMax Pro for Windows by selecting the Help menu in the menu bar Three types of help are immediately available you can view the contents of the help file seek specific help about formulas or get general information about how to use the help function a a Click here Contents for the Help Menu Using Help SoftMax Pro Help Options About SOF Tmax Pro Figure 1 3 Help Menu for SoftMax Pro for Windows Choosing Contents by clicking on the menu item with the mouse or pressing ALT H C brings up the SoftMax Pro help Contents window showing the gen eral organization of the help that is av
197. Figure 5 21 Group Settings Dialog Box for the Group Named Patient 1 The name sample descriptors and column format can be changed for the group that you are editing When you edit a group you will notice that the information shown for the Column Format in the Group Settings dialog box changes to show the name of the original group used to define the group columns see Figure 5 22 Group Settings Name Standards FD Sample Descriptor Concentration Units Imm F When you are editing Group Settings rather than creating a new group the word original precedes the Column Format Figure 5 22 Groups Settings Dialog Box Showing Original Column Format 5 25 Chapter 5 Reading Using Fluorescence or Luminescence Instruments 5 26 A ORGETONA Changing the column format setting will cause the columns in the group to revert to their original defaults as shown in Table 5 2 on page 5 24 If you have added columns or changed column information within the existing section for the group you are editing these changes will be removed The Button Allows you to deselect or clear wells in the map of the microplate Highlight the wells you wish to clear and then click the Clear button The Sample Area The Sample area contains the Sample drop down list and sample descriptor values active if defined in the Group Settings dialog box or if assigned auto matically by SoftMax Pro Sample 15ta01 e Sample Name or ID
198. Information about Enterprise Administrator 6 6 How Enterprise Administrator and the Enterprise Edition of SoftMax Pro Work Together 0 0 cece eee eee eee 6 6 InstallalOlEsesocewas ou repens dd dade tnded qi dite xu Ne 6 7 Administrator Login and Database lssleeeuss 6 8 a PEPPER 6 14 lgstuiu M 6 16 KEO e ad D SB casae eU PELIS dose tose s pius uds 6 18 Add SOW ALS ER 6 19 D lete 5OLDWOAEB uc dria dre Sem d ene Ha ADEL a CRO iem ee 6 19 Change PasswWOrd 424s ecarouesertesteredaddatnoumse gh 6 20 BU oe heen eeeeeraes 6 21 BOPO 6 23 POC TTA COTES 6 25 L166850 ETETETT dese PUPPI PET PRI SIONIS sa Ped dM 6 26 Enterprise Edition of SoftMax Pro 0 0 eee ee eee 6 28 DOCU In PLI 6 28 zinc MCCC 6 29 SoftMax Pro User s Manual vil Chapter 7 Displaying and Analyzing Data Using Absorbance InstrudmenEs oo vaio des wer ax nea ed iur osa 7 1 TIPO U CHOU TC 7 3 Initial Data Display and Reduction Plate CuvetteSet Section 7 4 TePe ee eee ee ree 7 5 The Display Dialog Box 1 2 0 eee eee eee ee 7 6 Graphing yyellSsses eumque eso sQed amen te aon terete ees 7 18 Masking Wells or Cuvettes 1 0 eee eee eee eee 7 22 Data ReQuchOn 2224ausds breues PERS d aS edd o RA ded es 7 23 lide suo Mm 7 23 COV Cle Se SOCOM eese otto tent rH deb IRE TS 7 23 ize EC 7 27 PIA E osos
199. IntensityLDLData txt Basic FP w o bkgrd subtraction pda Basic FP pda FPIA Assays w o bkgrd subtraction pda FPIA Assays pda G Factor Determination pda G Factor Determination pda LDL Determination pda Formula Reference Guide Examples Example 5 1 Method 1 pda Example 5 1 Method 2 pda Example 5 2 pda Example 5 3 pda Example 5 4 pda Example 5 5 pda Example 5 7 pda L tIest and Parallel Line Analysis Parallel lines pda tTest pda Absorbance Default Protocol ppr Drug Discovery Default Protocol ppr Endtut pda Gemini Default Protocol ppr Tutorial2 pda NOJ The data files for the tutorial are in pda Format You can access them by going to File gt Open and locating the Tutorial folder in your SoftMax Pro folder Only ppr files can be seen through the Assays menu 2 12 SoftMax Pro User s Manual Chapter 2 Installation Enter Registration Information SoftMax Pro User s Manual Turn the instrument on and allow it to complete its start up sequence approximately 30 90 seconds depending on the instrument type Then dou ble click the SoftMax Pro program icon UP inside the SoftMax Pro folder Mac or click the desktop icon or choose SoftMax Pro from the Start menu Windows to start the software program The first time you open SoftMax Pro a registration dialog box will appear asking that you enter your name company and the serial numbers of the software program and MDC Micro plate System instrument The softwa
200. Interpolate Raw Data Checkbox Top Raw Data without Interpolation Bottom Interpolated Data Custom Selected Showing Formula Button Endpoint suus Wavelength Calculation Dialog Box Endpoint 0 0 e eee eee Cursor Positioned on a Line between Columns SoftMax Pro User s Manual xviii Figure 8 26 Figure 8 27 Figure 8 28 Figure 8 29 Figure 8 30 Figure 8 31 Figure 8 32 Figure 8 33 Figure 8 34 Figure 8 35 Figure 8 36 Figure 8 37 Figure 8 38 Figure 8 39 Figure 8 40 Figure 8 41 Figure 8 42 Figure 8 43 Figure 8 44 Figure 8 45 Figure 8 46 Figure 8 47 Figure 8 48 Figure 8 49 Figure 8 50 Figure 8 51 Figure 8 52 Figure 9 1 Figure 9 2 Figure 9 3 Figure 9 4 Figure 9 5 Figure 9 6 Figure 9 7 Figure 9 8 Figure 9 9 Figure 9 10 Figure 9 11 Figure 9 12 Figure 9 13 Figure 9 14 Figure 9 15 Figure 9 16 Figure 9 17 Figure 9 18 Figure 10 1 Figure 10 2 Figure 10 3 Figure 10 4 Figure 10 5 Dragging a Column Divider Linie 4232235 4204 o9 esta 9E Ronde ies 8 40 The Show Formulas Command in the Group Menu 00 8 40 Formulas Shown in Group Columns 00 cess cece eens 8 41 Formula Button Activated and Column Formulas Shown 8 42 Column Formala Dialog DOX 25 33 9 24 ceded edet ed toda v4 v e pda eds 8 42 CaleataBom Digloe BOX 14 inest dome dte bon dre dorum picis E
201. MinStd Standards and Standards and Values lt Values Max MaxStd Standardsy Name MeanValue Result Formula Average Values InterpX STD Standard dardCurve Values Curve Values Std Dev Meankesult Stdev Values Average Result CV Std Dev Cv Values Stdev Result Cv Result Cv Result Dilution Std Standards I ur R Name Formula Name Formula Name Formula Name Formula Factor Name Adj Result Formula MeanResult Factor IConcentration and Factor are from SoftMax Pro version 1 x Concentra tion Factor and SampleDescriptor return identical information O1 24 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual The Button When you click the button the Group Settings dialog box will appear reflecting the settings for the active group currently visible in the group drop down list If you wish to edit a group that is not currently active first choose the group from the drop down list and then click the button The fig ure below shows the Group Settings dialog box for the active group called Patient 1 broup Settings Mame Patient 1 Name of Group Sample Descriptor VEJ Sample Descriptor Dilution Factor r Units for Sample Descriptor Column Format Setting Choices are Column Format nnn y Basic Standards Unknowns Unknowns Dilution
202. More than one text file can be imported into the same SoftMax Pro file by cre ating or copying another experiment into an existing file Analyst System files can be imported into a predefined protocol template in the Drug Discovery folder under Assays in SoftMax Pro All analysis features of SoftMax Pro can be used to analyze the file Import ELIPR Selecting Import FLIPR allows you to import FPD files that have been gen erated using FLIPR instrument control software version 2 0 and above Files are exported from the FLIPR software using the Plug Ins gt Import Export gt FLIPR import menu selection In order to import FLIPR files they must be exported by the FLIPR software into the specific FPD format Standard FLIPR files of the FID and FWD formats cannot be imported into SoftMax Pro Files can be imported into a predefined protocol template in the Drug Discovery 9 22 SoftMax Pro User s Manual Chapter 9 File Management and Printing folder under Assays in SoftMax Pro All analysis features of SoftMax Pro can be used to analyze the file SoftMax Pro User s Manual 9 23 Chapter 9 File Management and Printing 1 B Importing and Exporting Templates Template information saved as a tab delimited ASCII text file can be imported to or exported from Plate and or CuvetteSet sections A template file consists of seven columns of data separated by tabs Each line of the file provides information for one
203. New MaxValue Column Appended to Group Section View an If Statement in a Column Formula No conditional statements are used in the FLEX mode tutorial for the EC 50 analysis To view and learn about writing an if statement you will be asked to open a different data file If you do not wish to do this please skip this section and go on to Data Analysis Graph on page 10 64 Open the file named Endtut pda located in the Tutorial folder The Samples section of this file contains a columns called Outliers This col umn is made up of an If statement that calls upon data from another Group section to report when data points lie outside the range of the minimum and maximum Standard values An If statement in a formula defines a condition that if found to be true causes a certain result and if not true causes a different result The basic structure of an If statement is If condition resultl result2 Let s take a closer look at the individual elements that make up the Outliers formula If Values gt MinStd Standards and Values lt MaxStd Standards Outlier This statement says that if the data shown in the Values column is greater than or equal to the calculation of MinStd found in the Standards group section and less than or equal to the calculation of MaxStd found in the Standards Group section then show nothing signified by two quotation marks with nothing between them otherwise show t
204. On E Plate Last Read F oT L m p E No Data G o St Wavelength Combination Ler Data Mode Absorbance P i standards HeH ES Ern n p iii Controle Het Ey Ez a b FEE LInknowns uei 24 E ai b 7 Graph Fit b Cuvete Set iud Setup Template x Reduction Ei Display lke Al a Y HE H Figure 10 1 Default Protocol Tutorial 1 Notes and Plate 1 Sections Open SoftMax Pro User s Manual 10 7 Chapter 10 Tutorials L 3 If you are using an absorbance instrument CuvetteSet 1 shown in the absor bance file above bottom left can be deleted since the use of a cuvette is not covered in this tutorial To delete this section 1 Click in the title bar of the CuveteSet 1 section it will turn gray and the title will become bold From the Edit menu choose Delete CuvetteSet 1 A small dialog box will appear asking you to confirm deletion of this section Click the Delete button and the section will be deleted 2 The experiment should now have a Plate section Plate 1 a Graph section Graph 1 and two other group sections Controls and Unknowns that will be used in this tutorial 10 8 SoftMax Pro User s Manual t Chapter 10 Tutorials Step 2 Define the Instrument Settings Click the Plate 1 section to make it active its tool bar will become gray and then click the Setup button in the tool bar to open the Instrument Settings dialog box At the top of the dialog box
205. PET Ite 8 37 Displaying Data in Group Graph and Notes Sections 8 38 Group SECHIONS sasap dee ERNLATENLRIIU CE ELEME EAS NE Iis 8 38 INGLES SecHOflS scat cage ake Ge eee ee Is oo 8 45 Graph SecuOn ard Asante tue emu ede mea dex ata eee he 8 45 Formulas in 5oftMax Pro 22522 rz IR heb ol Ne y xb WESS 8 64 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 8 2 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Introduction SoftMax Pro User s Manual Initial display and reduction of the data received from the FlexStation instru ment is performed in the Plate section in which the data has been collected Display and reduction parameters can be set up either prior to or after collect ing data The display and reduction parameters may be modified at any time Data analysis and the display of analyzed data is also performed in the Group sections created when wells are assigned to groups in the Template Editor and the Graph section s Notes sections may be used in conjunction with any other section to provide text commentary and or a summation of data through the use of summaries Notes sections are used most commonly to provide an introduction to or sum mary of results for a data file All data files have a minimum of one Plate section with data in it Multiple Plate sections may be present Most files will have a tem
206. Plus or Plus will cause an error The Plate section will not display a reduced number and consequently no values will be reported in the Group section table 7 27 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 28 Kinetic Wavelength Combination The default wavelength combination for Kinetic readings is Lm1 and is the only choice besides Custom for all MDC Microplate System instruments except the SpectraMax and VersaMax For the SpectraMax the choices depend on the number of wavelengths chosen for example if you will read at two wavelengths choices default to Lm1 and Lm2 along with Custom With the VersaMax choices depend on whether you are reading the plate at either one or two wavelengths For any MDC Microplate System instrument if you do not wish to use the default reduction you can choose Custom and create a different reduction formula Kinetic Limits The Limits settings for the display of Kinetic data are MaxOD MinOD Lag Time and End Time The display of OD values is shown relative to the first point mea sured for each well Negative Kinetic values decrease with time and limits should be set accordingly below 0 to view negative Kinetic data Limits define the data that will be viewed and included in data reduction but do not affect data collection If you alter a limit to show less data you can always display the hidden data again by changing the limit MaxOD
207. Preferences Dialog Box Note that COM ports from 1 through 9 that are not available for your particular com puter will be shown dimmed A selection of None can be made if communication with an instrument is not required or if no instrument is connected to the computer When this option is selected the Status Bar in SoftMax Pro will show an instrument icon with an X through it along with the words No port selected This selection allows you to use the serial If you are using a Keyspan serial adapter with a Macintosh computer set the port to Printer SoftMax Pro User s Manual t Chapter 2 Installation ports for other purposes modem connection network connection or printing for example but does not allow you to communicate with the instrument NOH When the FlexStation is selected in the Preferences another option becomes available regarding the Serial Comm Speed see below mo Preferences Serial Port gt Autazawve File Export Format IFE Time Append Date v Include Labels File Prefi bas P Append Tine 2 Interpolate Wells 7 v SOPima PAO File Test File Add Filename and Date E gt Program Files SOF Tmax PRO 4 0 Reader FLEXstation ww Filters Autoprint Cancel Figure 2 9 Serial Port Speed Selection in Preferences Dialog Box FlexStation only SoftMax Pro automatically detects the baud rate of the instrument to whic
208. Print Setup dialog box for Windows and then repeat the print pro cess Additional information regarding the general print functions available in these dialog boxes can be found in the documentation that accompanies your computer and or printer Chapter 9 File Management and Printing Exporting Importing Information 9 16 Exporting Data The Export command a sub menu of Import Export located in the File menu creates a file of data from one or more Plate sections CuvetteSet sec tions and or Group table sections within a SoftMax Pro file The type of data that is exported depends upon three things 1 Selections made in the Preferences dialog box regarding the export format Choose Preferences from the Edit menu Export Format Selection Expart Farmat Ems Time v Include Labels Interpolate Wells AddFilename and Date Figure 9 12 Choosing an Export Format from the Preferences Dialog Box Note that the dialog box above shows preferences available for a FlexStation instrument with other instruments the Interpolate Wells function is dimmed not available 2 Within the Preferences dialog box that appears choose the settings for the data export that you prefer For Plate sections you can choose either time based format which pro duces a linear format for the data or plate based format which exports data in a matrix format corresponding to a microplate grid CuvetteSet sections a
209. RES 3 17 Croup SCCUION weosva hu 35 else Saale EE EE dius owe ea 3 19 Graphi Sec OIL eo pese tom qoa S Que RUE SERES ee ee 3 21 Instr ment Sell 8 uod paa diu nde Rei qos tp ERR 3 23 Menus and Commands erisia nri e TE envase SEA 3 23 APPIE M6eDU cei V EIREZNDASEERRUSUERP UR vis 3 26 About 5ott Max PIO s DR RP PEDEM Pern eer eet 3 26 Tale NIefitb o 28 5 aseo aun ames mone dde ee ee usc hte 3 26 Edit Men tocado E a Leder d wt ace wai PES 3 28 WAC Me 14 0 E R A ENA ciara esas te etal a eee 3 30 Experiment MENU sa viso ga o a Dee Ae penne es 3 30 COn WoL MENT sperd sve saos uso Beers IO dee selena es 3 31 ASSIS MEDU c heh pv telte ur UP eho returnees 3 32 Diste NVIeritis c ue acsi ese oe at ha SI ter in ease Ecce Sa S 3 33 uvettebet Menu wu Gores Moye beeen ca eee Pe aes 3 35 INGLES Ment oe dub a cute Bane des Beth dd adn Fae eee es 3 36 Grap k Ment ececheumeredsrsee dae ad edad uU M PS 3 37 Group Ment is ue 23 oy 35 else eee gee esa eiusd ses 3 38 Window Ment 54 65 8 aos cee Sk Bes Be EP PERDE ee MEE S 3 39 Help Menu Windows ce cee cee eee nnn 3 39 Help Menu Macintosh gy tau icebox e ere ee es 3 39 Interprocess Messaging i299 3 a dei sdan yeh RE EA EE a 3 40 Chapter 3 SoftMax Pro Overview t 3 2 SoftMax Pro User s Manual Introduction Chapter 3 SoftMax Pro Overview This chapter presents a general description of the features and use of SoftMax Pro It describes e the parts
210. RL P from the File menu You can determine which sections or whether all sections will be printed in this report see The Printed Report below 2 Acopy of the template can be printed while you are in the Template Ed itor by clicking the Print button in the bottom left corner 3 Areport can be printed automatically at the end of a reading To enable this function choose Preferences from the Edit menu and select Au toprint 4 You can print the active section or selection This option is available within the File menu If only one section is selected the name shown in the menu will reflect the name of the active section For example if the active section is named Plate 1 the topmost print command in the File menu would be Print Plate 1 If more than one section is select ed the menu will show Print Selection You can select multiple sections to be printed by holding down the Shift key and clicking on the section tool bars to make them active 5 Enlarged displays well graphs of Kinetic Spectrum or FLEX data can be printed by clicking the Print button in the Well Graph window The size of the printout is dependent on the size of the well graph If you enlarge the well graph to greater than 12 inches the printout will be truncated Regardless of which print option you select make sure the printer is con nected properly to the computer and that it is turned on prior to sending the print reque
211. Read You may choose to read the entire plate or only certain wells in the micro plate Partial plate reading is available for all read modes and the time required for some readings may be significantly reduced using this setting since the instrument does not have to read the entire plate To configure the settings for a partial plate read choose the Wells to Read set ting SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Highlight the wells to be read Wells must be contiguous but can be located any where in the microplate If you enable a partial plate reading only the wells to be read will be visible in the data display indicating that no data will be collected for the other wells all wells will be visible in the Template Editor however AutoRead Template Editor SoftMax Pro User s Manual This feature enables the automatic reading of subsequent Plate sections in the order in which they appear within an experiment You can set intervals delay time between the plate readings if desired The Template Editor is used to describe the location of samples in the wells of the microplate providing the link between the raw data and the analysis groups When you first open the Template Editor for a new Plate section the grid or display is empty The number of wells shown depends upon the type of plate selected in the Instrument Settings After selecting wells you can assign
212. SoftMax Pro User s Manual Version 4 3 Software for Macintosh and Windows c Molecular Devices Molecular Devices Corporation Sunnyvale California 94089 Part 4 5900 0328 Rev A Cc Molecular Devices Molecular Devices Corporation SoftMax Pro User s Manual COPYRIGHT Copyright 2002 Molecular Devices Corporation All rights reserved No part of this publication may be reproduced transmitted transcribed stored in a retrieval system or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without the prior written permission of Molecular Devices Corporation TRADEMARKS AND PATENTS FlexStation FLIPR SoftMax SpectraMax and Vmax are registered trademarks and Automix Emax Path Check ThermoMax UVmax and VersaMax are trademarks of Molecular Devices Corporation All other trademarks or registered trademarks are the property of their respective owners Automix is covered by U S Patent Number 5 112 134 PathCheck is covered by U S Patent Number 5 959 738 These trademarks are not to be used in any type of promotion or advertising without written permission from Molecular Devices Corporation DISCLAIMER Molecular Devices Corporation reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although t
213. SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Plates 1 Well Graph Raw 150 Time secs Yrax Points 34 well BS O D4 A ET Ymax 6 552 263 65 413 65 R Z 0 549 0 922 0 960 J Done Jr Figure 7 16 Well Graph for Three Kinetic Wells of a Microplate Raw Data The choices you make for viewing the data in the Plate or CuvetteSet section also affect the way the Well Graph appears Figures 7 14 and 7 16 show Well Graphs for raw data displays If you display one of the reduced options reduced number Threshold Ranged or Gray Scale the Well Graph will show the plot with an indicator of the reduced value and will include the word Reduced in the window title see Figure 7 15 which shows the Well Graph for a single Kinetic cuvette viewed in a reduced plots format Zoom Box You can enlarge the Well Graph even further by clicking the zoom box in the upper right hand corner of the window the center box of three in the upper right corner in Windows Clicking the zoom box once will cause the window to fill the total display on your monitor clicking the zoom box again will return the window to its previous size Size Box Clicking and dragging the size box located in the lower right corner Macintosh or clicking and dragging the lower right corner of the window Windows allows you to make the Well Graph any size desired Printing the Well Graph You can print the Well G
214. Standard amp Errors Wo Error Y Errors Mo Error Figure 7 38 Graph Options Dialog Box with Plot Name Changed to Standard 1 4 A second plot is added to this graph by clicking the New button in the Graph Options dialog box After clicking New a dialog labeled Plot 2 is opened The second plot name defaults to Plot 1 which now shows beneath the first plot Change this second plot name to Standard 2 choose Standard2 for the group and change the X and Y axes to show concentration and values respectively for Standard2 Graph Options Title Graph Height 100 Type onts MOS Scatter Sample Text PH itle L ah Cluster bar a Sample Text Set hi C Stack bar Legend J Connect points Sample Text Axes ES Plot symbols al Q Standard 1 O Standard amp Concentration Standard Y Malues Standard amp Errorz Ma Error Y Errorz Mo Error cancer Foe Figure 7 39 Graph Options Dialog Box with Second Plot Added for Standard 2 5 This process is repeated for a third plot labeled Standard 3 SoftMax Pro User s Manual 7 51 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 6 Now that all plots information is complete clicking in the Graph Option dialog box will close it and the resulting graph will show the three plots created XT 47 Graphite ite Fit Logoa w s 5 0 93 Grap
215. The Assays Menti consen ode hu bt beta peices oe eee dida we ied ftu edits da dapd a 9 3 Set Folder Dialog Box Macintosh 0 0 0 0 cece eee eee eens 9 3 Select Folder Dialog Box Windows 00sec cece eee eee 9 4 Save as Dialog Box Macintosh 0 0 cece cence 9 6 Save as Dialog Box Windows 0 ccc cece cee eee eee eee nnn 9 6 Preferences Dialog DOX icecsavetenverconangenmaneawan SE EE KEE EEEE ESEE 9 9 PENEDO DON era ae beeen A E E EE E EE iade dus adds 9 12 Moving a 5ecHon Upwards sosesesssrar aem an oirr ene kiena A P Roland 9 13 Format Text Dialog Box Macintosh 0 000 cece eet eens 9 14 Print Dialog Box for the Macintosh 0 cece eee eens 9 14 Print Dialog Box for Windows Epson Stylus 800 Shown 9 15 Choosing an Export Format from the Preferences Dialog Box 9 16 Ponar Dalt BOX esser sashes rebut aT A Vo dud he id ed er is 9 17 Export Dialog Box Windows and Macintosh 0 cee cece eee eee 9 18 Iuport aloe DOXGaaaver iode esae peti chai dua da dd eas aa D ardan aU a dus 9 22 Error Message Generated When Pasting Mismatched Data 9 26 Set Password Sheilding Dialog Box 0 eee cece eens 9 31 Password Required Dialog Box eee ccc eee een 9 32 Default Protocol Tutorial 1 Notes and Plate 1 Sections Open 10 7 Instrument Settings Dialog Box Showing Wavelengths Settings 10
216. Threshold Ranged Display PRG iss Sta Grayscale Ch Plot O1CeS Cancel Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 6 6 Data in the Plate section is displayed in a grid array microplate format With all plates other than 384 well plates the display is always shown as a stan dard microplate with well A1 in the top left corner If you are reading 384 well plates additional display options can be chosen see Figure 8 3 to make the display of data more clear e Normal shows the data in standard left to right format with well A1 in the upper left corner Columns 1 through 24 are along the top and rows A through P are shown from top to bottom The wells are smaller and data is shown with fewer digits les precision Vertical the plate is shown in the same orientation as Normal except that columns 1 through 12 all rows are shown above columns 13 through 24 all rows Rotated Same as the Normal view except that it is rotated 90 degrees clock wise well A1 is in the upper right corner Large Same as the Normal view except larger This display can be seen in its entirety only if you have the page size set for legal paper 8 5 x 14 inches in landscape orientation set this in Page Setup or Preferences under the File menu In the example in Figure 8 3 the display is shown as it appears if you do not change the page size from letter size to legal Interleaved
217. Tutorial after saving This does not mean the protocol has not been saved When you create a protocol file SoftMax Pro saves a copy of the current file under that file name but does not display the name on the screen Protocol files are saved as template files on the Macin tosh these are known as stationery files When you open a protocol file an Untitled window appears containing all the protocol infor mation saved previously so that you can collect data and save the resulting data file under a different name SoftMax Pro User s Manual 10 53 Chapter 10 Tutorials 10 54 Congratulations You have just created a SoftMax Pro protocol file Now would be the time to read the plate and then save the data file In this tutorial example however you will not perform an actual plate reading but will open a saved file to generate data that you will manipulate as if it had been obtained from an actual reading Step 7 Read the Plate A If you were reading an actual microplate at this time you would prepare the microplate as it is defined in the Template Editor Before an actual reading you might find it helpful to print a copy of the template to assist in properly filling the plate You would then place that microplate along with the appro priate compound transfer plate in the appropriate drawers of the FlexStation A new set of tips would also be loaded in the tip drawer Since we are using a saved file it is not necessary
218. Tutorial 1 Quantitative Endpoint Assay Tutorial 10 4 Hard and Soft Patamel6rsSs 2a nex ue OREL ede died 10 4 Description of the Assay Example 006 10 5 Define the Protocols iuo oaa dee S NAR ENORMES 10 6 Start SoftMax Pro Create a New File sees 10 6 Define the Instrument Settings 0 0 00 08 10 9 Define the Template 2 0 cece eee eee eee ees 10 12 Set the Reduction Parameters 00e cence 10 19 Set the Display Parameters 0000 esses 10 20 Read the Plale 25s visa edt oq use dites ete pidas 10 22 Data Analysis Group Sections 0 0 0 0 0008 10 25 Data Analysis Standard Curve 00 10 31 Pinta depot qe satan vat neat pee ee bade Pee eee 10 34 Tutorial 2 EC 50 Assay Tutorial FLEX mode only 10 35 Hard and Soft Parameters a edv atouts veas Keo oats 10 35 Description of the Assay Example 0206 10 36 Define the Protocol 20 e eee eee eee eens 10 37 Start SoftMax Pro Create a New File 00 0000 eee 10 37 Define the Instrument Settings 0 2000 10 38 Define the lemplates lt 25ic2deeectavdeteudesendeee dad 10 48 Set the Reduction Parameters 00 eee eee 10 51 Set the Display Parameters 0000 c eee eee ee 10 52 Read the Dale accurate n ed 10 54 Data Analysis Group Sections 0 000000 008 10 57 Data Analysis Graph
219. V on the Macintosh or pressing CTRL V in Windows USB Universal Serial Bus Port A serial communications port found on some Macintosh and PC computers An adapter may be required to connect your computer to this port see Chap ter 2 for more information User Name The User Name is the name of the person who reads data into the shielded file The User name is entered after the Read button is pressed but before the reading begins SoftMax Pro User s Manual B 15 Appendix B Glossary of Terms Vertical Display A display option for 384 well plates which shows the plate as two 8 x 12 arrays stacked vertically Vmax Points The number of points in the Kinetic or FLEX curve included in each of a series of regressions to calculate the maximum rate Vmax or the time segment used to calculate the Time to Vmax Vmax The rate reported as signal min milli OD RFU or RLU units per minute for a Kinetic or FLEX reading It is calculated using a linear curve fit y Ax B A creeping iteration is performed using Vmax Points and the slope of the steepest line segment is reported as Vmax Rate It may also be reported as units per second the default for fluorescence and luminescence Wavelength s Specifies the wavelength s that will be used to read the microplate Well Graph One or more wells in a plate section having data shown in plots format may be expanded by selecting them and clicking the Graph button on the Plate
220. Well Graph will be reduced data and the button will be set to Show Raw SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments e Scale to Data Scale to Limits button This button toggles between a view of the data within the reduction limits and a view of the entire data set Switching between these views helps to visualize how the limit settings affect data reduction If raw data is being viewed in the Plate section the display in the Well Graph is set initially to show the plot scaled to the reduction limits If reduced data is being viewed in the Plate section the Well Graph display is set initially to show the plot scaled to the data If you are viewing raw data and click the Scale to Limits or Scale to Data button only the display of raw data in the Well Graph will be affected the button will not affect the view of reduced data The same is true if you click this button while viewing reduced data To produce comparable scaling of both raw and reduced displays this button must be set to either Scale to Limits or Scale to Data for both raw and reduced views of the Well Graph Graph Options button Opens the Well Graph Options dialog box which allows you enable or dis able connected points or plotted symbols the graph Graph Options xf Connect Points kf Plot Symbols Figure 7 13 Graph Options Dialog X Axis Settings button Y Axis Settings button O
221. X4 R i Endpoint i s Patheheck constant Options 8 Water constant 3 Cuvette reference Wavelengths 450 poi PathChec 2 Automix amp Blanking Before Off Lint aso v Pre Read Plate Off j T You could preread the plate instead of using AutoCalibrate jme env SEA A A EE er a Pret i background constants On chal Above SpectraMax Plus ead entire plate P 384 V M PI AE us or VersaMax Plus Off GQ Automix amp Blanking feiss Auutamix dauesceesssccezsuscecessecuesscaeesseuscessscess ez eccctescceetesseeessest Before First Read SECS e T T Blanking WANABERRRREREERANEREANAANERENEEREREENRBANARNEREARSRERERNERNENANSEEANENNGEE Pre Read Plate AutoCalibrate Cocacacolcajcaicot oaa 2 Coacacocacacotoactaco V AutoCalibrate T OA cmn m r3 o m E Figure 4 1 Instrument Settings Dialog Boxes for an Endpoint Reading SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Kinetic Mode Wavelengths jf Number of Wavelengths 410 0 wk Run Time 5 00 meva 8 Number of Reads 34 Lint aso v i Minimum Interval 0 09 Maximum Interval 2 45 00 AAAUU A DDLDLLDDDDDD s m Instrument Settings Q Endpoint Automix amp Blanking E a rate Atomix EEA E E E S peor Z a 2 Before First Read secs 2 Wavelengths Wavelengths COO Steen r i ae Between Reads B ZELS i a O
222. a Standards pg ml Concentra MeanValue Std Dev CV After 072 Hiding 0 003 Replicates EE 0 004 6 5 0 001 17 7 Smallest standard value 0 004 Largest standard value 0 739 Figure 10 76 Standard Section Before and After Hide Replicates More information about modifying group sections can be found in Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using Close the Endtut pda file and return to the FLEX tutorial data file you opened earlier SoftMax Pro User s Manual 10 63 Chapter 10 Tutorials Step 9 Data Analysis Graph 10 64 Scroll down within the SoftMax Pro window until you see the section tool bar for Graph 1 If this section is closed click the triangle indicator on the left side of the tool bar to open it This graph contains a single plot as shown in Figure 10 77 EI Laramie vy Graph 1 1 Concentration y A DV x C 4B D A B C D Rza O Bot Data Concentration vs 96 Activati 64 926 0 533 0 007 102 161 0 999 Figure 10 77 Graph Section 4 Paramter Fit Applied This graph is set to show concentration on the X axis and Activation on the Y axis A 4 Parameter curve fit has been applied You can apply other fits by selecting one in the menu next to Fit Releasing the mouse button causes SoftMax Pro to redraw the graph based on its evaluation of the fit of the data to the new curve choice You can also display the
223. a for data files and can be opened only by the Enterprise Edition Files saved from other editions of Soft Max Pro ppr and pda files can be opened by the Enterprise Edition but will not have an audit trail associated with them General Information about Enterprise Administrator Enterprise Administrator software is designed to be used with various soft ware packages from Molecular Devices that have electronic signature and audit trail functions Enterprise Administrator software allows an administra tor to create a database in which users electronic signature and privilege information can be stored This database can then be used by other Molecular Devices software programs to validate the user s name and to retrieve associ ated privilege information This information determines the extent of the user s activities within the Molecular Devices software OXGYONA Molecular Devices has prepared some pre installation infor mation in this chapter and strongly suggests that you read it prior to using Enterprise Administrator software Enterprise Administrator has the following major functions SoftMax Pro User s Manual Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator User Information The administrator may add new users modify a user s properties and remove existing users After a new user is added that user can be allowed to use more than one Molecular Devices software program e Template A set of
224. a analysis and the display of analyzed data is also performed in the Group sections created when wells cuvettes are assigned to groups in the Template Editor and the Graph section s Notes sections may be used in conjunction with any other section to provide text commentary and or a display of data through the use of summaries Notes sections are used most commonly to provide an introduction to or sum mary of results for a data file All data files have a minimum of one Plate section or one CuvetteSet section with data in it Multiple Plate sections and or CuvetteSet sections may be present Most files will have a template and Group Graph and Notes sec tions However depending upon the assay and how you wish to report the data only some of these sections may be or need to be present in every file Some simple assays such as reading the absorbance of proteins at 280 nm in a cuvette or ina microplate with PathCheck allow you to calculate concentra tion in the Plate or CuvetteSet sections In such an example Group and Graph sections are necessary only if you wish to display the data graphically Many common assays consist of data acquired in a Plate section or CuvetteSet section Standards and unknowns are defined in a template Unknowns are then interpolated from a standard curve The following diagram shows the relationship between the different sections in SoftMax Pro Standard Group Table Reduced Number from Plate Cuvette
225. a shielded protocol file Notes e This command tells SoftMax Pro to save the protocol password internally so that when an OpenAssay command is issued using a shielded proto col SoftMax Pro will use this password to open the file SoftMax Pro User s Manual 12 13 Chapter 12 SoftMax Pro Remote Command Language e This protocol password remains in use until the program terminates or another protocol password is sent e The password is limited to 8 characters Example SetUserPassword bop i do Stop Send SoftMax Pro a command to stop reading If queue operation is enabled this command is not queued the queue is cleared and the command is pro cessed immediately This is similar to clicking the Stop button on the SoftMax Pro tool bar except the Stop button does not affect remote command process ing or its queue This command should be sent before any Close or Quit command if there is a possibility that the instrument is still reading NISENUNINB If the data has not been saved the application quits anyway with no user warning Visual Basic Excel Macro Example First some housekeeping code Private Declare Function FindWindow Lib user32 Alias _ FindWindow ByVal IpClaesName Ae String _ ByVal lpWindowName Ae String As Long Private Declare Function SendMessage Lib user32 Alias _ SendMessageA ByVal hWnd Ae Long ByVal wMeg As Long _ ByVal wFaram As Long _ ByVal IFaram As Stri
226. a template highlight the region and then select Copy from the Edit menu or press C Macintosh or CTRL C Windows Only the high lighted wells will be copied To paste the region into another area of a template highlight the region and select Paste from the Edit menu or press 3 V Macintosh or CTRL V Win dows Only the highlighted wells will have template information pasted into them Data is pasted using row priority all wells in a row of highlighted wells have SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments information pasted into them before proceeding to the next row from left to right If fewer wells are highlighted for pasting than were copied the data will be pasted until all paste highlighted wells are filled If more wells are highlighted for pasting than were highlighted for copying the extra wells are left empty SpectraMax Gemini fluorescence readers only If you try to paste template infor mation between Plate sections having different plate format settings an error will result Plates must be of the same type to copy and paste template infor mation Exporting and Importing Templates Templates can be exported or imported by opening the Plate section and choosing the appropriate command on the Plate or CuvetteSet menu ure Help uniri Help Template Template Wit 1398 Help anu Help Reduction Reduction New Cuvette New Cuv
227. ad area amp 1 H10 Compound Source Beckman 96 2 3mL Compound Transfer T1 100 1 30 17 Triturate T1 Not used Pipette Tips Layout Tip Area amp 1 H12 Compound amp Tip Columns T1 1 142 2 2 3 373 4 4 4 5 5 5 5 6 6 T 7 7 8 8 9 9 9 9 1010 10 on Cancel Figure 10 50 Instrument Settings Dialog Box Pipette Tips Layout Full Rack Chosen Compound amp Tip Columns These settings allow you to define the tips that will be used for the transfer of compounds that are also chosen here However this tutorial is not making any compound transfers so this setting is not used Final Check of Instrument Settings Instrument settings are now complete Verify that your settings agree with those shown below 10 46 SoftMax Pro User s Manual Chapter 10 Tutorials m Instrument Settings Orele Endpoint Kinetic Spectrum Well Scan Options Wavelengths Wavelengths Ex Em Auto Cutoff On 495 525 515 p Number of Wavelengths Seer nee A ER SoC COT On Sensitivity ee Ree Readings 3 Excitation Emission Iv Auto Cutoff Dm um Bes Te Es Tel Timing Time 200 secs Interval 2 Secs Reads 101 Automix Before Off Between Ott AutoCalibrate Once Assay Plate Type 96 Vell Standard clrbtm Wells To Read Read area A1 H10 Compound Source Beckman 96 2 3mL Cancel Compound Transfer Mane Triturate n a Pipette Tips Layout Tip Area amp 1 H12 Compo
228. ailable Help Softmax Pro Help Window File Edit Bookmark Help Menu Softmax Pro Help The SOFImaxz Pro Window Contents of the Heading a Microplate SoftMax Pro er Help File Templates Status Bar Data Display Reports Figure 1 4 Help Contents for SoftMax Pro for Windows 1 10 SoftMax Pro User s Manual Chapter 1 Welcome to SoftMax Pro Help Options from the Contents Window e You can search for a particular item or subject by clicking the Search but ton located in the tool bar at the top of the Contents window Type a word or select one from the list Then choose Show Topics Enter the subject you are searching for in this box Select a topic then choose Go To Figure 1 5 Using the Search Function within Help in SoftMax Pro for Windows After entering a search subject clicking the Show Topics button will cause a list of related topics to appear in the box below me Search Type a word or select one from the list Then choose Show Topics Formulas Formulas Fract Function Getting Started Graph Menu Graph Section Select a topic then choose Go To Edit Column Edit Summary Choose a Formula Help tovic from Group Menu nee list Recalculate Now Reduction Show Formulas Figure 1 6 Choose a Topic within Help in SoftMax Pro for Windows e You can return to the window viewed previously by clicking the Back button e Clicking the History butt
229. ailable Items above the gray line are custom groups that may be created as you wish The Group drop down list will appear differently depending upon the cus tom groups that have been created by default or by the user The figure below shows two different instances of the appearance of the Group list Patients Standards my Unknowns my Blank Blank Hew Mew LLL LLLILLA No custom Example of custom groups created groups created by a user Figure 5 19 The Group Drop Down List Depending on the default template used when opening SoftMax Pro initially no groups may appear above the gray line As you create new groups they will be positioned above the line e Blank Creates a plate blank in the Plate section Note the name blank is reserved for use by the SoftMax Pro program and should not be used when creating custom groups e New Creates a new group The Group Settings dialog box will appear see Figure 5 20 showing default settings This dialog box allows you to define the name for a group of related samples what type of descriptor will be associated with these samples and the initial column format for the Group section Whenever a new group is created whether or not wells are selected in the template a Group section table will also be created To delete a group you must delete the Group section Simply clearing a group from the Template Editor will remove the assignment of wells to that group name but
230. alibrate For absorbance instruments an air reference reading is taken before runs and sometimes between Kinetic readings during a run as determined by the type of instrument and read settings For fluorescence instruments measurement occurs before reads and sometimes between Kinetic reads if the interval is long enough Calibration data is stored in volatile memory and is used by SoftMax Pro until the instrument is powered down or the wavelength is changed Autocalibration can be turned off see AutoCalibrate on page 5 18 for more information Automix The Automix function determines how often if at all automated shaking of the microplate is performed during a reading This feature is covered by U S Patent Number 5 112 134 AutoRead This feature enables automatic reading of subsequent Plate sections in the order in which they appear within an experiment You can set intervals delay times between the plate readings if desired Baud A data transmission rate measured in bits second for communication devices Blanking CuvetteSet Section SpectraMax Plus or Plus only Three types of blanking are available in the CuvetteSet section for the Spectra Max Plus or Plus 9 Ref Blanking Template Blanking and Group Blanking A Ref Blank is created when you click the Ref button or choose Ref from the Control menu This command causes the SpectraMax Plus or Plus to read the cuvette in the cuvette port and appli
231. ally but if you choose to do this the information will not appear automatically in the Group section To see it you will need to create a new column containing the formula ISampledescriptor For more information about creating columns see Adding Columns on page 7 45 Column Format defines the default column types that will be created for a new group or shows the original column format setting that was used to cre ate the group you are editing Four selections are offered Basic Standard Unknowns and Unknowns Dilution Different types of columns will be created for a new group or changed in an existing group depending on which of these column format settings you select Table 4 2 shows the default columns created with each selection type NOH If you are creating a new group for unknowns that have a dilution factor use the Unknowns dilution column format as a base rather than the Unknowns group included in the default protocol 4 25 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments zi e weed Hx No C Q weed 3 o Q a h n a SQ a a S 3 wee un ISampleNames Im Concentration Concentration Values R Outside Standard Range Name Formula SampleNames Name Wells Formula WellIDs Name Sample Formula Index Name Values WellValues WellValues If Values gt MinStd Standards and Values Max Std Standards 44 it Mf
232. alog Box Macintosh Save this document az Save In E Protocols File name Tutorial ppr Save as type Pro Protocol Files ppr Cancel Figure 10 21 Save As Dialog Box Windows On the Macintosh click the option button next to Protocol File In Win dows click in the box under Save File as Type and choose Pro Protocol Files ppr Type the name you are giving to the file in the text box high lighted over Untitled In this example type Tutorial When you have fin ished entering information in this dialog box click to save a copy of the protocol file to the hard disk The title bar of the window will still show Untitled rather than Tutorial after saving This does not mean the protocol has not been saved When you create a protocol file SoftMax Pro saves a copy of the current file under that file name but does not display the name on the screen Protocol files are saved as template files on the Macintosh these are known as stationery files When you open a protocol file an 10 21 Chapter 10 Tutorials Untitled window appears containing all the protocol information saved previously so that you can collect data and save the resulting data file under a different name Congratulations You have just created a SoftMax Pro protocol file Now would be the time to read the plate and then save the data file In this tutorial example however you will not perform an actual plat
233. alog Box Choose the file you wish to import and click the Import button Note that data to be imported must contain the appropriate header information A new experiment is created in the open SoftMax Pro file for the imported data New Plate sections and or CuvetteSet sections are created for each data set within the file Plate pre read and CuvetteSet reference data can also be imported but data for path length correction cannot Note that the time date stamp for a section contain ing imported data shows imported data rather than a date or time Import Analyst Choosing Import Analyst allows you to import data files generated by Ana lyst System instruments including Analyst AD HT Acquest and ScreenSta tion Files for import must be generated as verbose reports using Criterion Host 2 0 or SCREENPLAY 2 5 for all modes ABS FI FP Epi ABS Lum and the dual method saved as text txt When importing 1536 well data from the Analyst System instrument only the first quadrant of 1536 well plate data the top left section of 24 by 16 wells can be imported This data will be displayed in the standard 384 well plate format Instrument parameters for each plate in the text file will be imported into a Notes section as text and the raw data will be imported into a Plate section The plate ID from the text file will be used to name both the Plate and the Notes sections and the file name will be used to name the Experiment
234. alog box associated with the column formula will open allowing you to edit the formula and the name associated with the column SoftMax Pro User s Manual 6 41 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 1 k 8 42 Create Summary Button Create Column Button Formula Button i Stdew v ali CM ar D 104 41 2 Smallest standard value MinfMeanvalue 0 252 Largest standard value Max Meanvalue 0 252 Figure 8 29 Formula Button Activated and Column Formulas Shown For a complete discussion see the accompanying Formula Reference Guide Adding Columns You can add a new column to a Group section by clicking the Create Column button in the Group tool bar or by choosing Create Column from the Group menu which will open the Column Formula dialog box Calculation Namej toif Decimal Places Figure 8 30 Column Formula Dialog Box In the Column Formula dialog box you can change the name of the new col umn from the default which simply lists the column by number to reflect the data that it will contain If no existing column is selected when you click the Create Column button the new column will be created to the right of all existing ones If you select highlight a column and then add a new column it will be positioned to the right of the one you selected If the table is currently as wide as the sheet of paper
235. alysis formulas add columns containing new formulas and or add one or more summaries at the bottom of the section Graph sections are used to plot information from groups defined in the Template Editor Values associated with the X or Y axes depend on the type of group se lected for graphing More about Graph sections can be found in Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using General Information about Sections 3 10 Sections are either active or inactive When active the section title bar is col ored dark gray Multiple sections can be active at the same time The tool bars of inactive sections are color coded to make it easier to discern a section s type Inactive Experiment or Section Color Pastel Shade EXPENDED oru nied ence Debbie d Gray eee re re ee ee er PUES Purple CU Velle Tous qoo qoth e d eerie anes Green hor DER Yellow OUD rr Pink Conn PETER Blue Clicking anywhere in an inactive section makes it active Selecting an experi ment deactivates any individual section within it that may have been active and vice versa Activating a section also activates the experiment containing that section To activate multiple sections at the same time hold down the Shift key and click in the tool bars of the sections you wish to activate Doing this allows you to move copy duplicate print and delete multiple sections simultaneously The section item in the SoftMa
236. an be found in the fol lowing section Wavelengths The FlexStation instrument automatically reads up through the bottom of clear bottom microplates Wavelengths Choose the number of different wavelengths to be read At least one wavelength must always be specified Selecting more than one wavelength will cause a corresponding number of wavelength setting boxes to appear maximum four wavelengths SoftMax Pro User s Manual 5 41 Chapter 5 Reading Using Fluorescence or Luminescence Instruments i Wavelengths Number of Wavelengths 2 we Excitation Emission Auto Cuttaff cum fos I Ee Jr 55 om fe dm pm de 59 Figure 5 38 Wavelength Settings The wavelengths specified previously or the defaults will be shown in the boxes You can choose a wavelength from the choices in the pull down menu or simply type any wavelength within the available range of the scanning monochromator in the FlexStation instrument over an existing setting The pull down menu will show common monochromator settings Sensitivity Readings This setting determines the number of readings that will be performed on each well of the microplate readings are averaged and the average reading is displayed r Readings a rU Readings ar ERE EE Fast Normal Precise Fast Mormal Precise Figure 5 39 Readings Setting The illustration on the left shows the default setting for fluorescence the right shows changing this
237. and 8 FLEX A read mode using fluorescence that is available with the FlexStation instru ment You can specify compound additions and trituration for individual wells Data is collected over time with readings taken at regular intervals that can be less than one second FLEX runs are measured in seconds and fractions of seconds FLEX readings can be single or multiple wavelength up to four SoftMax Pro User s Manual Appendix B Glossary of Terms FLIPR Molecular Devices FLIPR Fluorometric Imaging Plate Reader system was developed to perform cell based high throughput screening HTS assays Fluorescence The light emitted by certain substances resulting from the absorption of inci dent radiation To measure fluorescence accurately it is necessary to reduce light scatter The governing equation for fluorescence is Fluorescence extinction coefficient concentration quantum yield excita tion intensity pathlength emission collection efficiency Fluorophore A material that absorbs light energy of a characteristic wavelength under goes an electronic state change and instantaneously emits light of a longer wavelength Four Parameter Logistic Curve Fit The equation used to generate this curve fit is y A D 1 x CB D More information regarding curve fits can be found in Chapters 7 and 8 Gain The amount of increase in signal power expressed as the ratio of output to input for a ph
238. and FlexStation instruments clicking the button that shows a thermometer causes the Incubator dialog box to appear allowing you to set and regulate the temperature of the mi croplate cuvette chamber The temperature setting can be left at the default or can be specified by typing a different value in the box The incubator setting is independent of the protocol being run Run ning an assay does not automatically set the temperature set point After a reading however the temperature set point range and aver age temperature are recorded in the saved file The Automix button Clicking this button shakes the microplate Note This is a manual shaking of the plate as opposed to an automatic shaking that can be chosen in the instrument settings and is not enabled for the Emax Drawer button Clicking this button opens or closes the microplate drawer NOINA The status bar can be hidden from view by selecting Hide Status from the View menu Selecting Show Status from the view menu will restore it to view Experiments and Sections Experiments Each SoftMax Pro file contains at least one experiment and can contain more than one Within an experiment are one or more sections discussed below Experiments define a naming scope for the sections within them and provide quick access to different types of information without the need to open or refer to multiple files An experiment can be selected by clicking its tool bar the ar
239. any subsequent slopes are calculated beginning at the second time point and ending at a total number of readings equal to Vmax Points The steepest slope is reported as Vmax Rate If the data plot displays fewer time points data points than the number of time points chosen for Vmax Points then all of the time points will be used to determine the slope of the data 6 26 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual Table 8 1 Using Vmax Points Example Time points D aaeoa in the well data plot Number of Points Used for Data Analysis Vmax Points set to 25 25 default Vmax Points set to 5 25 Overlapping Overlapping 5 point line 5 point line segments segments SoftMax Pro will perform a linear regression on as many overlapping 5 point line segments as possible in the analysis window The slope will be determined for each line segment and then the steepest slope will be reported as the reaction rate Thus in well 2 of the example in Table 8 1 above the slope will be determined for line segments consisting of time points 1 to 5 2 to 6 3 to 7 4 to 8 5 to 9 and 6 to 10 and then the slope of the line segment with the steepest slope will be reported as the Vmax Rate The progressive slope calculations are repeated until the last included data point is reached as defined by the End Time or MaxRFU MaxRLU see bel
240. aph will not fit on an 8 5 by 11 inch page and the printout will be trun cated SoftMax Pro User s Manual 8 17 6 18 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Masking Wells If you find data in certain wells outliers that should be excluded from the calculations to be performed by data reduction you can mask these wells Select the well s to be masked and then click the Mask button in the Plate section tool bar or choose Mask from the Plate menu Masked wells are shown with an overlay of diagonal lines The masking function can be used as a what if tool For example you could suppress or enable certain functional groups within the experiment Suppose you have included a group blank in the template and want to see the data with and without the blank Masking the group blank wells would sup press the blanking function unmasking them would enable it again v CG Plated i bs m per sec Md CII Le dem Ee nterra r23 Reads 14 ee al eal L 7 cu Masked SSS EEE iim A A udo cuan Wells Wavelength Combination Lrm1 E Autamix Ott Calibrate On PhT Medium Feagdsziell 5 Lag Time 0 00 End Time 5 00 RFU Min 0 RFU Max 20000 Wimax Pla 14 14 Plate Last Read 4 25 PR 2 5 2001 Figure 8 12 Masked Wells in a Plate Section To remove masking from one or more wells highlight the masked well s and click the Mask button in t
241. appear to lie on or scattered around a straight line This equation is shown on the screen as y A Bx A linear fit should be used whenever the standard values appear to lie on or are scattered around a straight line Figure Figure 8 40 shows an example of a linear fit a mer Y Standard Curve qi a m m 2 z 30 Concentration y Bx o Std Standards Concentration ws Meanvalue 0 058 Figure 8 40 Graph with Linear Fit SoftMax Pro User s Manual 6 53 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Semi Log The semi log function fits the best straight line to a set of data for log X plot ted against Y The resulting curve displayed will be a straight line with the X axis drawn in logarithmic scale The equation for a semi log fit is Me B n log49 X and is shown on the screen as y A B log x where A is the y intercept of the line and B is the slope note log in this equa tion is the common or base 10 logarithm we 2 Standard Curve Fit Standard Curve ph x m Cc wm DE E Concentration J A B Logis o Std Standards Concentration ws PTean alue 0 254 Figure 8 41 Graph with Semi Log Fit 8 54 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Log Log The log log function fits the best straight line to the set of data which consists
242. arameter that is not found in the Instrument Settings dialog box SoftMax Pro User s Manual 5 5 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Endpoint Mode Wavelengths f Number of Wavelengths v Fluorescence RFUS Excitation Emission IV Auto Cutoff Luminescence RLUs Li Bes v Ex v Time Resolved RFUS Sensitivity M Instrument Settings xj B Readings Fast Normal Precise e CO INMME D TIRE EUN r7 PMT Sensitivity OO Read Type Fluorescence Wavelengths Ex Em Auto Cutoff On 485 538 530 Sensitivity i Readings 6 i Time Resolvecenre PMT Auto Automix Before Off AutoCalibrate AutoCalibrate On Assay Plate Type 96 Vell Standard Wells To Read Read entire PRN e TEA E E ee de t uertit ecce tum d AutoRead Off nOD Automix Fluorescence RFUs C Luminescence RLUs Assay Plate Type Wells To Read Select an assay plate type 96 well plates 96 Vell Standard es 384 well plates 384 Well Standard 48 well plates 48 Vell Costar 24 well plates 24 Well Costar 12 well plates 12 Well Costar 6 well plates See 6 Well Costar Turn autoread on Delay b secs Figure 5 1 Instrument Settings Dialog Boxes for Endpoint Mode 5 6 SoftMax Pro User s Manual Chapter 5 Rea
243. ard value Max Meanvalue 0 253 Figure 7 31 Formulas Shown in Group Columns Modifying Column Formulas To modify the formula for an existing column double click the column or click the column once and then click the formula button in the section tool bar The dialog box associated with the column formula will open allowing you to edit the formula and the name associated with the column Create Summary Button Create Column Button Formula Button eT ill Standards MeanValue Average V aluez Standards mg ml Concentration Wells Values Std Dev CIS I5ample Concentration welll weis al i Stdeee V alu Cv Val O 000 B1 0 122 0 104 41 2 B2 0 135 C 0 143 ce 0 165 D 0 182 0 201 0 223 0 246 0 272 0 2300 0 332 0 367 0 406 0 448 Smallest standard value MinfMeanvalue 0 253 Largest standard value Max MeanValue 0 253 Figure 7 32 Formula Button Activated and Column Formulas Shown For a complete discussion see the accompanying Formula Reference Guide 7 44 SoftMax Pro User s Manual L d Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Adding Columns SoftMax Pro User s Manual You can add a new column to a Group section by clicking the Create Column button in the Group tool bar or by choosing Create Column from the Group menu which will open the Column Formula dialog box Calculation Namej terf Decimal Places Figure 7 33
244. arning on your own Information about the tuto rial files is found in Chapter 10 Note that folder and filenames are the same for Windows and Macintosh except that Windows files have the extension ppr or pda appended to the file name for protocol files or data files respectively Enterprise Edition files use extensions epr and eda All of the protocol files that are on the software CD are installed as shown in the table below and can be seen in the Assays menu within SoftMax Pro Table 2 1 Files that Are Installed Basic Protocols All Kinetic ODs for Export ppr Basic Endpoint Protocol ppr Basic Kinetic Protocol ppr Basic Luminescence ppr Basic Pathcheck Protocol ppr Basic Percent Binding ppr Basic Percent Control ppr Basic 5pectrum ppr Basic TRF Protocol ppr Raw Optical Density ppr Cytoproliferation CyQUANT Fluorescence ppr Drug Discovery Basic FP w o bkgrd subtraction ppr Analyst Basic FP ppr not installed with FPIA Assays w o bkgrd subtraction ppr FPIA Assays ppr G Factor Determination ppr LDL Determination ppr Life Sciences version SoftMax Pro User s Manual Chapter 2 Installation Table 2 1 Files that Are Installed Drug Discovery FLIPR EC50 percent ppr Inot installed with EC50 and 2factor ppr Life Sciences version Endpt ELISA AP and pNPP ppr Bgalactosidase w ONPG ppr HRP and ABTS w Acid Stop ppr HRP and ABTS w SDS Stop ppr HRP and OPD w Acid Stop ppr HRP and TMB ppr Urea
245. assic mode e 17 inch monitor e 256 MB RAM 6 GB hard disk drive e CD ROM DVD ROM drive e Extended keyboard e USB to Serial Adapter and adapter software e Epson 740i color printer Minimum System Configuration e Apple Power Macintosh G3 e MacOS 8 6 e 17 inch SVGA monitor e 128 MB RAM 2 GB hard disk drive e CD ROM drive Either configuration above requires a straight through serial cable either an 8 pin DIN to 8 pin DIN for SpectraMax VersaMax and FlexStation or 8 pin DIN to DB 25 cable for Emax Vmax UVmax and ThermoMax Molecular Devices recommends using only high quality double shielded straight through serial cables If you plan to use SoftMax Pro with a FlexStation instrument or with any instrument if you will be creating large files in excess of 20 plates per file Molecular Devices recommends that you increase the amount of RAM in your computer to a minimum of 256 MB 2 The minimum configuration may not provide enough memory for creating or working with large files more than one plate or cuvette per file and or the use of robotics If your computer freezes or stops functioning try increasing the amount of memory allocated to the SoftMax Pro program see Chapter 11 for more information 2 6 SoftMax Pro User s Manual Chapter 2 Installation eee HN Windows Compatible Computer Windows Recommended System Configuration e Pentium IV 1 8 GHz processor e Microsoft Windows 2000 e 17 inch
246. ate Editor is to delete the corresponding Group section from the experiment SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Selecting Wells in the Template Editor In the Template Editor for a Plate section you can select one or more wells by clicking or dragging on the grid Multiple contiguous wells can be selected by dragging across them Non contiguous selections can be made by holding down the Shift key and clicking individual wells Clicking a row label will select the entire row clicking a column label will select the entire column Contiguous columns or rows 1 12 or A H for a 96 well plate for example can also be selected by dragging Non contiguous columns or rows can be selected by holding the 3 command key on the Macintosh or the CTRL key in Windows and clicking in labels Double clicking anywhere within a group will select the entire group To deselect all wells quickly click in the empty border around the Template e When you leave the Template Editor by clicking the button the Plate section will show the boundaries of the groups created in the tem plate Group labels and sample descriptors will not be visible you can see them by holding down the Ctrl Shift keys When you release the key s the original view of the Plate section will return The Template Editor Tool Bar SoftMax Pro User s Manual The tool bar will appear differently depending upon what
247. ate Sum mary button in the tool bar or choose Create Summary from the Group menu The Calculation dialog box will appear allowing you to change the name of the summary or hide the name if desired as well as to enter a description of the summary and to create a formula for it including the number of decimal places for the result if appropriate Double clicking an existing summary causes the same dialog box to appear allowing you to modify summary infor mation Calculation Name Summary O Wide Name Decimal Places Figure 8 31 Calculation Dialog Box Information entered in this dialog box will be placed at the bottom of the Group section as follows Name Description Formula Result You can click the checkbox next to Hide Name which will change the way the summary appears to show Description Formula Result NOJ The name assigned to a summary is important since you can refer ence the summary by name in other formulas building one upon another The summary name can be referenced even if it is hidden If you wish to see the formulas associated with summaries without opening the Calculation dialog box for each one you can choose Show Formulas from the Group menu To see these formulas only briefly hold down the shift and control Ctrl keys while that Group section is active The summaries will expand to show the complete formula along with the title They will resume their previous appearance when you release the optio
248. ate a custom reduction formula to better suit your needs Since all custom reduction formulas are defined the same way regardless of read mode they are described together after the individual read mode sections Wavelength Combination Kinetic The default reductions for Endpoint readings are Lm1 and Custom for all fluorescence instruments when you read at a single wavelength Additional wavelength combinations become available depending on the number of wavelengths chosen You can choose Custom and create different reduction formulas The wavelength combinations available in the drop down menu show the correct format of the formulas that produce these reductions Wavelength Combination The default wavelength combination for Kinetic readings is Lm1 Other selections depend on the number of wavelengths chosen for example if you will read at two wavelengths choices default to Lm1 and Lm2 along with Custom If you do not wish to use the default reduction you can choose Cus tom and create a different reduction formula Kinetic Limits SoftMax Pro User s Manual The Limits settings for the display of Kinetic data are MaxRFU RLU Min RFU RLU Lag Time and End Time The display of RFU RLU values is shown relative to the first point measured for each well Negative Kinetic values decrease with time and limits should be set accordingly below 0 to view negative Kinetic data Limits define the data that will be viewed and incl
249. atement dialog box SoftMax Pro User s Manual 6 19 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator T Sien Statement j Requesting approval for the current experiment Disagree Add 4 Note Figure 6 13 Sign Statement Dialog Box Disagree Cancels the signature process and closes the dialog box Add A Note Allows the user to add a note to the statement prior to signing it Agree Prompts the user for a his her password and then upon proper entry signs the document with the user name date and time Once a statement is signed the Experiment will be locked and can not be edited unless signatures are removed Unsigned statements may be signed but no futher changes can be made Multiple signa tures may appear on one statement Signatures are saved with data files but are not saved with protocols kl statements a Approval Requesting approval far the current experiment Note added when signed Experiment approved Signed by Charles Lexington on Tue Jun 18 2002 17 37 Figure 6 14 Siened Statement Section 6 20 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Introd to HOT State SA jee ESO M dirt deas Ou oa d 7 3 Initial Data Display and Reduction Plate CuvetteSet Section 7 4 The Data d5pl3y 5e zo de d Dos Eye Up eor spot Se de 7 5 The Display Dialog BOX2 ses ucro etx a ue CHER RE SER we 7 6 Graphing Welles te
250. ation Lm1 Data Mode Absorbance C Wavelength Combination Lm1 Data Mode Absorbance D Figure 7 6 Resizing Cuvettes in the CuvetteSet Section A Cuvette at original size B When the mouse pointer is positioned over bottom line of cuvette display it changes to show a crosshair cursor with vertical arrows C Dragging the cursor down shows the bounding box displaying where the boundary for the cuvette will be when the mouse is released D Resizing a cuvette display is different from enlarging the cuvette to a graph by double clicking the cuvette or clicking the Graph button or choosing Graph from the CuvetteSet menu which shows an enlarged display of either Kinetic or Spectrum data in a separate window Resizing the cuvette in the CuvetteSet section provides an enlarged view of the data in the same win dow note also that the resizing is saved with the file so the modification to the size will be retained when you reopen the file at a later time Figures 7 7 Plate section and 7 8 CuvetteSet section show the default dis plays for Endpoint Kinetic and Spectrum modes The default display for Endpoint mode is raw optical density collected from the microplate reader For Kinetic and Spectrum modes the default display is raw OD shown in plot format OD vs time for Kinetics and OD vs wavelength for Spectrum scans Data for most instruments may also be viewed as T Transmittance instead of OD optical density by ch
251. ations SoftMax Pro 4 0 4 01 Update Specification GLP GMP Support New levels of file protection have been added e In addition to standard password protection of a file you may now add an author s name to a file This name is saved with the file will print with the report and is shown on the screen To prevent any and allchanges to a file including saving it under a different name or accidentally overwriting it with a file of the same name you may choose to shield a file The author of the file must include his her name and password as well as a new shielding name and password as part of the file protection e Exported files now show the date author and file name e All printed pages now show the file name date author and Molecular Devices copyright information on all pages Analyst System Files e Import Analyst has been added to the Import menu This selection allows you to import verbose reports generated using Criterion Host or SCREEN PLAY software for all instrument modes After import the data from the Analyst System file can be viewed and ana lyzed just as if it had been generated by SoftMax Pro FLIPR Files e Import FLIPR has been added to the Import menu This selection allows you to import FPD files exported from FLIPR software After import the data from the FLIPR file can be viewed and analyzed just as if it had been generated by SoftMax Pro Robotics e Autosaving a file now saves time
252. ationship 7 3 8 3 tool bar 3 11 Security Menu 6 28 Select All command 3 28 Semi Log curve fit 7 55 7 56 8 53 8 54 B 13 Sensitivity PMT 5 43 Serial port 2 16 11 10 B 13 G4 2 4 iMac 2 4 Series 4 30 4 31 5 27 5 29 replicates 4 30 5 27 Shielding 9 29 Show All command 3 38 7 42 8 39 Show Formulas command 3 37 3 38 7 43 7 46 7 47 8 40 8 44 8 45 Sign Statement 6 32 Signatures 6 32 Size box 7 21 8 17 Slope 7 35 8 28 8 33 B 14 Smoothing 8 33 SoftMax files 9 6 8 45 8 52 8 52 SoftMax Pro User s Manual SoftMax Pro earlier version files 9 6 What s New 1 4 Specifications Macintosh computer requirements 2 6 Windows compatible computer 2 7 Spectral emission scan B 6 Spectral excitation scan B 6 Spectrum 1 3 4 11 5 12 7 36 8 29 B 14 display defaults 7 6 8 6 Speed Read 4 13 Standard curve 7 64 8 61 10 31 Standards minimum number 7 64 8 61 Start and End wavelengths 7 36 8 29 Statement 6 29 Stationery file 10 21 10 53 Status bar 3 4 3 7 4 43 5 35 5 52 Stokes shift B 14 Strips 4 18 5 18 Summary 7 46 7 47 8 44 8 45 adding editing 7 41 7 46 8 38 8 44 aligning 7 46 8 44 formula 7 46 8 44 line up with column 7 46 8 44 moving 7 46 8 44 plot 7 47 8 45 Suspend Recalculation command 3 29 7 40 8 38 T Temperature display 3 7 Template 3 9 4 22 5 21 blanks 4 34 clear 9 26 B 3 command 3 33 3 35 copy paste 4 35 5 33 9 25 groups 4 20 4 21 5 19 5 20 hierarchy 4
253. ay terminate the License by returning to MDC all Software documentation the original Software all backup and archival copies of the Software and all demonstration copies of the Software The License agreement will terminate automatically without no tice from MDC if the User fails to comply with any provision of this License Agreement It is incumbent upon the User to take all reasonable measures to effect termination only in accordance with this License Agreement GENERAL 1 This License Agreement shall be governed by the laws of the State of California 2 The provisions of this License Agreement are severable and if any one or more of such provisions are judicially determined to be illegal or otherwise unenforceable whether in whole or in part those remaining provisions or portions of the License Agreement unaffected by such determination shall be binding on and enforceable by and between the User and MDC 3 In the event it becomes necessary to retain the services of an attorney to enforce any provisions of this License Agreement the non prevailing party to such legal action and or litigation agrees to pay the pre vailing party s costs including reasonable attorney s fees and court costs if any 4 The User acknowledges that by opening the media package the User has read this License Agreement understands it and agrees to be bound by its terms and conditions The User further agrees that this License Agreement constitutes the full and c
254. bar File Edit View Experiment Control Assays Graph Help File Edit Miew Experiment Control Assays Window Help Figure 3 15 Menu Bar Macintosh top Windows bottom While working with SoftMax Pro you may access its tools and features through several means menus tool bars dialog boxes and keystrokes Fig ure 3 16 shows all of the menu items that are available in SoftMax Pro When menu items are unavailable they are dimmed shown as gray rather than black available choices depend on the action you are performing with the software An ellipsis three dots following a menu item indicates that choosing this item will bring up a dialog box Menu items followed by a trian gle are hierarchical menus choosing such an item causes a submenu to appear to the right of the original item Certain menu items can also be chosen by pressing keys on the keyboard Within Windows menu items contain an underlined letter in their names Pressing the ALT key will activate the menu bar pressing a letter key that corresponds to the underlined letter of a menu name will then activate that menu Subsequent menu items can be activated chosen by simply pressing the underlined letter in their names you do not need to press ALT again For example to open a file from within SoftMax Pro using the keyboard shortcut you would press ALT F O which will first activate the menu bar then select the File menu then choose Open from the File menu Whe
255. bars Clicking these buttons accom plishes many of the same actions as choosing menu items 3 24 SoftMax Pro User s Manual LA Chapter 3 SoftMax Pro Overview Menu Dimmed Keyboard About SOFTmax PRO Title Command Shortcuts File New Open Close Minimize ae M Expand a6 E Hide Status Experiment 1 P Notes 1 Copy Paste Clear Save Save As Import Export Experimentz2 P Plates Export Select All Set Password CuvetteSets1 Pa Import Criterion Delete Plate 1 Je Eup Duplicate Platest1 Print Standard Curve Import FLIPR p Ellipsis Print 36P ext Style ET indicates that Quit 0 Preferences choosing this command will Suspend Recalculation open a dialog box Recalculate Now 35z Experiment Control New Experiment Instrument Setup set Folder Template Reduction New Notes Read 3R protocol 1 New Plate GK Ref Display New Cuvetteset Close Drawer Graph New Graph Incubator Mask Calibration Copy Template Open Tip Drawer Dacta Tan only shown i lemplati Open Compound Drawer when FlexStation Set Air Gap isa Export Template Recover Import Template Section Name New Window Create Column Mew Cuvette Create Summary Create Summary Edit Summary Graph Options X AXIS Y Axis Template Show Formulas Reduction N
256. be set before the plate is read and can be changed after the plate is read The data display will update automatically each time The Vmax Points setting does not affect data collection The height of the Y axis in the raw data plots or plate display is con trolled using MinRFU and MaxRFU The default settings are MinRFU 0 MaxRFU 20 000 The width of the X axis in the raw data plots or plate display is con trolled using Lag Time and End Time If either the MaxRFU or End Time setting excludes points such that a total number of readings equal to Vmax Points cannot be used then the maximum included points will be used Time to Vmax This is an alternative method for analyzing non linear FLEX readings that reports the elapsed time until the maximum reaction rate is reached rather than reporting the maximum rate itself Used in conjunction with Vmax Points Time to Vmax is the time to the midpoint of the line defined by Vmax Points and used to calculate Vmax This elapsed time data is useful for appli cations including coagulation chemistry where the changing concentration of the reagents does not change Vmax but rather will change the time at which the reaction reaches the maximum rate Onset Time This is another method for analyzing non linear FLEX readings Onset Time reports the time required for a FLEX reading to change by a specified amount onset RFU above the initial RFU reading for the well Time at Minimum This setting rep
257. box might look like at this point depending upon which instrument is connected to your PC wa Instrument Settings d xj Endpoint Kinetic Spectrum Options Wavelengths 450 PathCheck e Number of Wavelengths 1 wb Li x Automix amp Blanking KJ Iz EP Before Off Lmt fs0_ v es M Pre Read Plate Off Quis ele AutoCalibrate jp m RRiae On Strips Options Read Type Fluorescence p Number of Wavelengths i Read entire plate AutoRead Ex Em Auto Cutoff On i f Off 485 538 530 i Excitation Emission IV Auto Cuttoff Readings 5 PMT Auto Automix i AutoCalibrate Sensitivity un les v xs w 530 v Cancel 96 Well Standard Wells To Read Read entire plate Figure 10 2 Instrument Settings Dialog Box Showing Wavelengths Settings Top left absorbance instrument bottom right fluorescence instrument Sensitivity Fluorescence instruments only Now check the settings for Sensitivity by clicking that heading on the left side of the dialog box the defaults shown on the right side 6 Readings Automatic PMT Sensitivity are fine and do not need to be changed for this tutorial Automix or Automix and Blanking Now check the settings for Automix fluorescence or Automix amp Blanking absorbance by clicking the heading on the left side of the dialog box on the right Automix Before should be set to Off
258. but these last parameters may be set up and or modified after data collection SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments 1 2 3 4 5 6 7 8 9 10 11 A typical process for preparing collecting and analyzing data is as follows Start SoftMax Pro Create Plate section s as needed Define instrument settings use buttons in the Plate section tool bar or the Control menu Define the template reduction parameters and display parameters Prepare the microplate s to be read and place them in the instrument Prepare any additional compounds to be used and load the required pipette tips Initiate the reading Save the data file Modify the template reduction parameters or display parameters as you wish or if you didn t set them up in Step 4 do so now Save the data file Analyze the data using the Group section tables and Graph sections Save the data file when you are finished SoftMax Pro User s Manual 5 37 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Instrument Settings NOJA Only the settings that pertain to the instrument that is connected to the computer or the one chosen in the Preferences will be shown in the Instrument Settings dialog box You must specify the instrument settings prior to actually reading a micro plate These settings include e The read mode FLEX e Timing e PMT sensitivity and
259. buttons and pull down menus Buttons in the tool bar are only enabled when that section is active the section does not need to be open Menu equivalents exist for all tool bar functions Figure 3 5 shows the active tool bar from a Graph section D or Fit Eger F Figure 3 5 Tool Bar from an Active Graph Section If you scroll the window vertically so that part of an open active section is hidden its tool bar sticks to the top of the window see Figure 3 6 This fea ture prevents the active tool bar from becoming unavailable if you are view ing the bottom of a section that is longer than the size of the window When the next section scrolls to the top of the window its tool bar replaces the pre vious one but is not active even if the section is open until you click within it If you scroll back so that even a small part of the previous active section is visible its tool bar returns to the top of the window File Edit View Experiment Control Assays Plate Help eaa 4 on be data m gt Active v Eg Pite Cutoff Tool Bar fe AA AA LA EmsStart Stop AAAALAAALUAA ALAR ALLA sux Active Open Afi iin bann then bin Automix Off Section AAA LENA s LA LEA ARLE i Scrolling E Reads well amp Wavelength Combination Lrod Upward Plate Elank Used Start 350nm End 7S nm RFU Min 0 RFU Max 20000 Flate Last Read 8 42 aM 6 20 22 Inactive x Standards Tool Bar
260. c tion SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments AutoRead Template Editor This feature enables the automatic reading of subsequent Plate sections in the order in which they appear within an experiment You can set intervals delay time between the plate readings if desired NOES Use of the Template Editor with the FlexStation instrument is the same regardless of read mode and is described in the previous chap ter on page 5 19 and following Reading a Microplate SoftMax Pro User s Manual The FlexStation instrument sends fluorescence data to SoftMax Pro Fluores cence is defined as the measurement of light that is emitted after absorption or excitation from radiant activation of a compound You can start a reading at any time after defining instrument settings It is not necessary to define groups and assign wells within the Template Editor first The values received from the instrument are raw absorbance fluorescence luminescence or time resolved fluorescence readings and are not affected by settings in the Template Editor Only instrument settings must be defined prior to reading a microplate To read a microplate open the microplate drawer of the instrument click the drawer button in the status bar or choose Open Drawer from the Control menu and insert the prepared microplate Then simply click the Read but ton in the status bar or choose Read from t
261. ccount the mechanical speed of the pipetter head the time needed to aspirate and dispense fluids trituration and Automixing The minimum time for the second pipetting event depends on when the first pipetting event occurred The calculation for the second event starts at the end of the first event and adds to that the total time that will be nec essary to perform actions prior to aspirating new fluids from the com pound plate and dispense them into the assay plate The Compound Transfer setting works in conjunction with the Compound amp Tip Columns setting described below Until well assignments are made in the Compound amp Tip Columns setting the Compound Transfer setting will show No targets assigned Triturate SoftMax Pro User s Manual The choices shown in this section are derived from the number of compound transfers specified If no transfers are enabled no triturate settings are possible This setting is used to enable a mixing of the contents of the wells in either or both the compound source and assay plates You can set the volume of fluid to be withdrawn from the well and the number of times cycles it will be pipetted back into that well With the Assay Plate setting you are also asked to enter a value for the height at which the transfer occurs The height setting should take into account the amount of fluid currently in the well in order for the pipette tip to either remain out of contact with the flui
262. ce luminescence value expected most or all of the data plots should appear in the data display In the example shown in Figure 8 16 if the MaxRLU value is increased from 10 000 to 20 000 the plots then appear as shown in Figure 8 18 v C3 Plater a set ll Z2 a Vinax units per sec Plate 1 3 3 4 4 7 _ 4 1 M 13 A ie Kinetic Time 5 00 JE L7 Interval 0 09 C f Reads 34 D Fi l pe i p du p Uu Fluorescence E 4 E LA L Bottom read FLA LA ZYE YY Ex Em Cutoff a L L V4 7 Lm1 485 538 530 H Aito Cutoff Wavelength Combination Lm1 Automix Off Calibrate On PMT Medium Reads Well 6 Lag Time 0 00 End Time 5 00 RFU Min 0 RFU Max 20000 Vmax Pts 34 34 Plate Last Read 10 27 AM 1 22 2001 Figure 8 18 Kinetic Reduction with Absolute Values Enabled and MaxRFU Value Increased SoftMax Pro User s Manual 8 25 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Kinetic Reduction Kinetic reductions are applied to the list of numbers the value at each time point after the wavelength combination formula is applied Vmax per min and Vmax per sec reductions are available for all instruments that are capable of Kinetics the default Kinetic reduction is Vmax units per sec Other reduc tions available from the pop up menu are Time to Vmax Onset Time Time at Minimum Ti
263. cence Instruments FLEX SoftMax Pro User s Manual Vmax The maximum slope of the FLEX plot of relative fluorescenceunits versus time The default value displayed is in relative fluorescence units RFU per second The rate is calculated with the use of Vmax Points see below which determines the number of contiguous points over which Vmax is calculated The number of Vmax Points defaults to the maximum taken during the read ing for all instruments except the FlexStation which defaults to 5 points You can change this number in the Reduction dialog box Vmax Vmax sec e 60 000 A linear regression is performed to determine the slope of the line using the equation y m X B where m is the slope of the line The reaction rate is reported in RFU sec Vmax Points This setting defines the maximum size of the line segment used to determine the slope of the line used in calculating the rate of the reaction The default is the total number of points taken in the reading with the data normalized to a starting point of 0 0 as opposed to starting from the optical density of the first time point The first slope is calculated for a line drawn beginning at the first reading as defined by Lag Time described below and ending at a total num ber of readings equal to the Vmax Points setting The second and any subse quent slopes are calculated beginning at the second time point and ending at a total number of readings equal to Vmax Poin
264. choices in the pull down menu or simply type any wavelength within the available range of the scanning monochromator in the instrument over an existing setting The pull down menu will show common monochromator settings SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments For Spectrum Readings When Spectrum is selected the Wavelengths section of the dialog box changes to request a wavelength scanning range and a wavelength increment Step between the start and stop settings see Figure 5 10 Wavelengths Excitation Emission Cutoff so w see Stop 750 Figure 5 10 Wavelength Selection for a Spectrum Reading The minimum selectable increment is 1 nm For fluorescence and time resolved fluorescence readings you must also choose whether to read excitation at a fixed wavelength and scan emission wavelengths or vice versa within the range of the instrument 250 850 nm A procedure to optimize excitation and emission wavelengths for a given assay is outlined in the instrument manuals for the SpectraMax Gemini fluo rescence readers and FlexStation instruments Please see those manuals for more information When luminescence is chosen you must also select a start and end wave length and a step minimum step increment is 1 nm Cutoffs SoftMax Pro User s Manual The choices in this portion of the Wavelengths setting depend upon the selec tions for read mode Endpoin
265. chronousl X Clipboard Figure 12 1 SoftMax Pro Remote Command Interface Notes On Queued Processing SoftMax Pro User s Manual To enable queued processing specify R for Remote in the program com mand line when launching SoftMax Pro This option is optional in order to maintain compatibility with previous versions that did not include queued processing After sending a block of remote commands the caller should wait until the commands have been completed by sending the command ReturnStatus which returns the number of remote commands in the queue yet to be exe cuted When ReturnStatus returns the message that there are zero remote commands waiting the caller should then check that the reader is idle using the Status command When the queue is empty and the reader is idle the caller may clear the command queue and send another block of commands The command queue is limited only by the memory capacity of the com puter No SoftMax Pro error will be generated when the user has flooded the software with remote commands which results in overloading memory 12 3 Chapter 12 SoftMax Pro Remote Command Language A special command has been created to clear the queue ClearQueue This command is processed immediately See SoftMax Pro Commands on page 12 9 for more details The Stop command is not queued and processed immediately See Soft Max Pro Commands on page 12 9 for more details Send Receive Remote
266. cked Figure 11 3 SoftMax Pro Info Dialog Box The actual dialog box will show a version number Enter a larger amount of memory in the box next to Suggested size the amount you enter may be limited by the total amount of memory available The next time you start SoftMax Pro it will use the larger memory size you have allocated You may also enter a different number into either the mini mum or preferred size boxes It is suggested that you do not reduce the num ber in the Minimum size box you may increase it however The Preferred size can be as large as you wish depending on the amount of memory available SoftMax Pro User s Manual 11 17 Chapter 11 Troubleshooting 1 3 HW T 11 18 General Problem 2 1 2 3 4 5 6 Printshop Launches instead of SoftMax Pro Windows only Both Printshop an application from Brederbund and SoftMax Pro use the extension pda to identify files If Printshop is already installed when you install SoftMax Pro on your computer the pda extension will have already been registered with Windows as a Printshop file and SoftMax Pro files will appear with the Printshop icon Double clicking any file ending with the pda extension will launch Printshop instead of SoftMax Pro There are two ways to fix this problem 1 you can launch the SoftMax Pro program and open the data or protocol file from within the program 2 you can change the program that starts when you
267. cted using Kinetic mode not generally rec ommended a pre read with the same Kinetic read interval and duration as the normal reading will be done and the ODs at each time point of the pre read will be subtracted from the OD of the corresponding time point of the normal reading 4 19 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 1 k Template Editor 4 20 Molecular Devices recommends that the wells of the pre read plate be filled with the appropriate buffer for the assay or with water when performing pathlength correction The Template Editor is available for Plate and CuvetteSet sections In both cases it is used to describe the location of samples either in the wells of the microplate or in a cuvette providing the link between the raw data and the analysis groups The Template Editor is used to create a template which pro vides SoftMax Pro with a map of what is in the microplates and or cuvettes When you first open the Template Editor for a new Plate section or CuvetteSet section the grid or display is empty After selecting wells or cuvettes you can assign groups and indicate the type of samples they will contain Depending upon the default protocol you are using certain group names and associated sample descriptions may have been created You can use these predefined groups and or create new groups to suit your needs When creating new groups you may enter any name you wish Once e
268. ctions are first created the formulas used to calculate the data are not displayed These formulas can be shown until you choose to hide them again by mak ing a Group section active and then choosing Show Formulas from the Group menu Note that the command in the menu changes to Hide Formulas after they are shown Create Column Create Summary Edit Summary Hide Replicates Show Formulas Show Formulas Command Group Settings Mew Window Figure 8 27 The Show Formulas Command in the Group Menu SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Choosing this option causes an additional row to appear beneath the head ing for each column which shows the formula used to calculate the data in each column To see formulas briefly hold down the shift and control Ctrl keys Standards mg ml Sample Concentration Wels Values MeanValue Std Dev CV Formulas i ICancentration well IL eTa Avera e ValuiStdev Vall CrtVal Smallest standard value Min Meanvalue 0 253 Largest standard value Max Meanvalue 0 253 Figure 8 28 Formulas Shown in Group Columns Modifying Column Formulas To modify the formula for an existing column double click the column or click the column once and then click the formula button in the section tool bar The di
269. curve fits can be found in Chapters 7 and 8 Luminescence The emission of light by processes that derive energy from essentially non thermal changes the motion of subatomic particles or the excitation of an atomic system by radiation Mask Lets you hide the data in selected wells of the Plate display so that the data stored there will not be used for calculations and will not be reported The Mask function is commonly used to suppress outliers Maximum Reduction for a Spectrum scan that reports the maximum signal within the reduction parameters MaxOD MaxRFU or MaxRLU The limit for the maximum value you wish to use for displaying and analyz ing Kinetic data or Spectrum scan data The default is 100 OD or 20 000 RFU or RLU Any values that are above these limits will not be shown and will be excluded from data reduction H Maximize Button Windows The small box containing an up arrow at the right of the title bar Mouse users can click the Maximize button to enlarge a window to its maximum size Key board users can use the Maximize command on the Control menu Menu A list of items most of which are commands Menu item names appear in the menu bar at the top of the window To choose an item in a menu place the pointer on the menu heading and drag to the selection you desire then release the mouse button Menu Bar A list of items most of which are commands Menu item names appear in the menu bar the horizonta
270. d Cuvettes Using Absorbance Instruments Dual wavelength is represented by Lm1 Ref because the subtraction of the second wavelength from the first takes place in the firmware of these instru ments causing a single number to be reported by SoftMax Pro For Spectrum Readings SpectraMax Instruments When Spectrum is selected the Wavelengths section of the dialog box changes to request a wavelength scanning range and a wavelength increment Step between the start and stop settings see Figure 4 10 The minimum selectable increment is 1 nm Figure 4 10 Wavelength Selection for a Spectrum Reading Speed Read SpectraMax Plus Plus 190 and 340PC only Speed Read performs a fast Spectrum scan of Plate sections or CuvetteSet sections Speed Read does not measure the total amount of light available at each wavelength Ip but uses the values stored in NVRAM of the instrument As a result Speed Read may not provide as accurate an absorbance measure ment of each wavelength in the scan as with the normal mode Speed Read Figure 4 11 Speed Read Section of the Instrument Settings Dialog Box SoftMax Pro User s Manual 4 13 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments k PathCheck Pathlength Correction SpectraMax Plus Plus 9 190 and 340PC only The Instrument Settings dialog box for the SpectraMax Plus Plus 190 and 340PC includes a section for PathCheck pathlength correction
271. d also include blank wells within different groups The effects of the different blank ing functions are cumulative so it is important to understand the function of each before using more than one type of blanking in a single assay Pre read blanking plate blanking Ref and template blanking are applied to the raw data acquired from the instrument whereas group blanks are applied to the reduced number Group blank subtraction is only visible in the Plate sec tion CuvetteSet section when the reduced number is displayed Figure 4 31 shows the last two types of blanks in the Template Editor 4 34 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Experiment 1 Plate 1 von ee ED seen E s us Clear 6 7 8 5 1D 11 T2N hic Ma Lu t 1 Thos RUE TR02 TE TR 01 TR O1 TROI TROI TROZ TROZ TROZ TROS TRO BL Plate Blank aE l Group Associated Blank Wells E N o AAA RR TR 01 TR Di TRU Rot TRO2 TROZ TRO TR OZ pepe Ror ro ma TE mete me uin TR o2 TROZ TR z TR 03 TR 0z o TRO TRO TR 02 TR OZ TROOZ TR 03 TR 03 TR 01 mo TRO TRO TRO2 TF OZ TRO TR OB TR OZ TR 03 TR 01 TROT TR OH Trot TR O02 TR 02 TRO2 TROZ TR Oz TR OZ Figure 4 31 Template Editor Showing Plate Blank and Group Associated Blank Wells In the figure above note that the plate blank in the upper left appears as a separate group named Blan
272. d in the well to avoid cross contamination or to engage the fluid vO Transfer 1 Transfer 2 ew Compound Sollee eje Assay Plate e Volume Hoo oo ful v aluma so i ace Cycles EE Height Hon H Figure 5 46 Triturate Settings Box 5 47 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Pipette Tips Layout Molecular Devices recommends that you always use a full rack of tips the default If you use less than a full rack of tips it is imperative that you change this setting to match the correct number and positions of the actual tips inserted in the tips drawer see the Caution below Figure 5 47 Pipette Tips Layout Settings Box If you change the Assay Plate Type setting from a 96 well plate type or vice versa after making selections in the Pipette Tips Layout tab the selection of pipette tips will need to be redone as it will no longer match The Assay Plate setting takes precedence over all other fluidics module settings A ONORA Molecular Devices recommends that you use a full rack of tips each time you perform a transfer of fluid in FLEX mode since this prevents potentially serious errors from occurring when partial tip racks are used For example if you should mistakenly enable a pipet ting function from a tip that is not present or if you enable more or fewer tips than are actually available the instrument can malfunc tion potentially causing serious damage While it is po
273. dary emission to produce enough electrons to generate a useful current 5 16 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments En Automatic E PT Sensitivity RARE High Automatic wr Ped Low Figure 5 13 PMT Sensitivity Setting SpectraMax Gemini fluorescence readers Clicking in the box causes a menu of choices to appear Automatic is present only when Endpoint mode is chosen Otherwise settings are manual and limited to High Medi um or Low The voltage of the photomultiplier tube may be set to low for samples having a higher concentration medium or high samples having lower concentra tion in all read modes In Endpoint mode and also Spectrum mode when using the SpectraMax Gemini XS or EM an additional setting automatic is available This setting allows the instrument to adjust the PMT voltage auto matically for varying concentrations of sample in the plate Automix The Automix function is a patented feature that allows you to set automated shaking of the microplate The options available for Automix depend on the read mode chosen and the type of instrument connected or chosen in the Preferences Automix pee Automix E Automix L Before First Read 5 sees Before First Read i D Between Reads z secs Figure 5 14 Automix Settings Box Before First Read This setting can be used with any read mode Click the checkbox
274. data New Group section tables can also be created but are devoid of any data New Graph sections include all settings for plots fit etc but contain no data New Notes sections are exact copies of the active Notes section including any summaries and all text same as duplicated Notes sections except for the name Empty Notes section Replicates Instrument Setup Reduction and Display parameters from last Plate section created if no Plate section exists default values are used Replicates Instrument Setup Reduction and Display parameters from last Cuvette Set section created if no CuvetteSet sec tion exists default values are used Creates a new Graph section with default settings and asks for plot information Not available Replicates text and summaries from source Notes section Replicates Instrument Setup Reduction and Display parameters from last Plate section cre ated Replicates Instrument Setup Reduction and Display parameters from last CuvetteSet sec tion created Also copies the last reference that was read and the number of cuvettes defined from the source CuvetteSet section Replicates all Graph settings including any plots that were defined in the source Graph section Creates a replicate of the source Group section table The new group will be in the template Group drop down menu but no wells will be assigned to this group SoftMax Pro User s Manual 1 3 Chapter 9 File
275. de the SpectraMax Plus 9 and the SpectraMax Gemini XS The calculations for large well plates have also been updated Autosave Changes The Autosave option in the Preferences dialog has been enhanced so that the file creation date can be appended to the file name when files are saved in sequence An Append Date checkbox is used to activate this feature The names of the files created when Append Date is activated begin with the file prefix followed by the date the iteration number and the file extension If the file prefix field is left blank in the Preferences dialog the autosaved files will use the protocol creation date as the file prefix Autoprint Changes When Autoprint and Autosave are both selected in the Preferences dialog the name used in the footer of the printed document will match the name of the correspondingly saved file rather than the on screen document name If Auto print is used without Autosave the on screen document name is used SoftMax Pro User s Manual A 5 Appendix A SoftMax Pro Version Specifications SoftMax Pro 3 1 1 Update Specification Correction of Problems in SoftMax Pro 3 1 Several problems were found in SoftMax Pro 3 1 that were corrected in Soft Max Pro 3 1 1 e Password protected files from version 3 0 did not open correctly in version 3 1 e In the Display dialog displaying raw data in 1000s could not be deactivated after it was activated e Well scan files with custom
276. der Filters Autoprint Cox Figure 9 6 Preferences Dialog Box When you enable the Autosave function by clicking its checkbox in the Pref erences dialog box the textbox below it becomes active allowing you to accept or change the defaults for saving data files The default location is the folder containing the SoftMax Pro program You may accept or change the default file prefix and file type and you may choose to append the date and or time after the prefix as part of the saved file name The prefix for an Autosaved file can be a maximum of 15 characters This lim itation is imposed in order to ensure that the addition of either or both time and date do not extend the length of the file name beyond 31 characters stan dardized at this number because it is the maximum allowable length for the 9 9 Chapter 9 File Management and Printing 1 B 9 10 Macintosh for more information regarding the maximum length of file names on the PC see the NOTE on page 9 9 The file type for Autosaved files can be either SoftMax Pro file or ASCII text file If you select the Text file option you can then choose either the Create a new file or Append to file option You may choose to append the date and or time to the autosaved file name Appending the date will include the date after the prefix e g Data 8 14 01 Choosing to include the time after the prefix will append the time that the file was saved in hhmmss
277. des instrument control and analysis functions for all SpectraMax and earlier instruments The Drug Discovery version provides instrument control and analysis functions for the FlexStation instrument as well as import and analysis of data files from FLIPR and Analyst instruments The Enterprise Edition includes all features from both the Life Sciences and Drug Discovery versions and also contains features that enable 21 CFR Part 11 compliance Only an administrator may install the Enterprise Edition of Soft Max Pro and this should be done after installing the Enterprise Administrator software program The administrator must enable cer tain functions within SoftMax Pro that link these two programs together and will have the required information about all users of SoftMax Pro Enterprise Edition in order to do so If you are an admin istrator and need to learn more about installing these programs you should first read the installation instructions in the manual that accompanies the Enterprise Administrator software Then return to this section and read the rest of the installation instructions for the Enterprise Edition If the administraor password to a database is lost the data within that database will be unretrievable Administrators may wish to develop a procedure to safeguard their user name and password to prevent alteration and or data loss Follow these instructions to install SoftMax Pro from the CD to your com puter s hard disk
278. dialog box allowing you to start or stop temperature regulation and to select an elevated temperature for the microplate chamber on instruments that have temperature control capability Instrument Icon Located in the status bar the instrument icon shows the status of the connec tion between the computer and the instrument If the icon has an X through it the connection is not functioning or no instrument is connected if the instrument icon appears without an X the connection is working properly Instrument Setup Defines the parameters mode wavelengths automatic mixing run time read interval etc used to read the microplate or cuvette Interleaved Display A display choice in which the wells are shown in a format that skips every other well as follows all odd columns and rows begin in the upper left corner of the plate display and are followed by all even columns and rows This dis play is most useful when the 384 well plate comprises four daughter plates of 96 wells each Interprocess Messaging Windows only Messages that can be sent to and received from other applications This fea ture is very useful for interfacing MDC Microplate System instrument with robotic systems and for sending and receiving data from LIMS systems See Chapter Chapter 12 of this manual for information about the commands that are available and how to use them Kinetic During Kinetic readings data is collected over time with multiple rea
279. ding the status bar will show the actions that are occur ring in the MDC Microplate System instrument along with a box showing the elapsed time for each action in minutes and seconds The Read button will change to Stop allowing you to terminate a reading if necessary An exam ple of the appearance of the status bar at the start of a Kinetic reading of a microplate using a SpectraMax instrument is shown below Reading 7 of 24 Figure 4 41 Status Bar during a Kinetic Reading SpectraMax ml 0 00 04 ad Plate 4 43 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments k Calibration SpectraMax Plus Plus 9 190 340PC and VersaMax When the instrument is connected to a SpectraMax Plus Plus 94 190 340PC or VersaMax or when one of those instruments is chosen in the Reader pref erences a Calibration command is available in the Control menu Choosing this command opens the Calibration dialog box Either a Plate section a CuvetteSet section or both can be selected for calibration The dialog box also shows the date of the most recent calibration Calibration C Calibrate Plate Last Calibration 1 01 1900 SpectraMax O Calibrate Cuvette Last Calibration 5 12 1955 Plus Plus Calibration Calibrate Plate Last Calibration 1 01 1900 SpectraMax 190 and 340PC Figure 4 42 Calibration Dialog Box SpectraMax Plus or Plus and 190 340PC Instrument calibration per
280. ding Using Fluorescence or Luminescence Instruments Kinetic Mode Wavelengths r Number of Wavelengths CS cca i Excitation Emission IV Auto Cutoft Fluorescence RFUs Lm kes v Exe v C Luminescence RLUs C Time Resolved RFUs Fast Normal Precise j f PMT Sensitivity M0000000510000000000000000008 500000999 m Instrument Settin Js x ej T MM i pectrum Well Scan Run Time 5 00 Read Type Fluorescence Interval 3 Wavelengths G i Number of Reads 34 Ex Em Auto Cutoff On E ae a Minimum Interval 0 36 485 538 530 Maximum Interval 2 45 00 Sensitivity i Readings 6 i C Time Resolved RFUs PMT Medium i Timing Automix Time 5 00 Interval 0 22 Reads 14 Minimum Interval 0 15 i Before First Read sacs Automix Between Reads secs Before Off emm RSS Between Off AutoCalibrate Cancel On AutoCalibrate Assay Plate Type 96 Well Standard Wells To Read Read entire plate AutoRead Off C Lumineseeer e TALUS c Automix IDMNIMMRIMUIMUMUTUU UEM Assay Plate Type Wells To Read Select an assay plate type 96 well plates 96 Yell Standard 384 well plates 384 Well Standard 48 well plates 48 Well Costar 24 well plates 24 Well Costar 12 well plates 12 Well Costar 6 well plates La nNmMmMo xS 6 Well Costar AutoRead uy
281. dings made at regular intervals measured in seconds The values calculated based on raw Kinetic data are Vmax Vmax per Sec Time to Vmax and Onset Time Kinetic readings can be single or multiple wavelength readings up to six if using a SpectraMax instrument other than the SpectraMax Gemini or Gemini XS which have a maximum of four or the FlexStation which also has four Lag lime Lag time in a Kinetic assay is the period of very slow growth microorgan isms or the rate of reaction that may precede the rapid linear phase of reac tion When a lag time is specified in SoftMax Pro data collected prior to the lag time is not included in data reduction Large Display A display choice available with 384 well plates This setting is useful when looking at large numbers if you are using a monitor that is 17 inches or larger Linear Curve Fit The linear function fits the best straight line to the data The equation for this fit has the form of y A Bx SoftMax Pro User s Manual Appendix B Glossary of Terms where A is the y intercept of the line and B is the slope A linear fit should be used whenever the values appear to lie on or scattered around a straight line More information regarding curve fits can be found in Chapters 7 and 8 Log Logit Curve Fit The Log Logit is also called a two parameter curve fit The equation used to generate this curve fit is y A D 1 X C D More information regarding
282. disappears 1 From the View menu choose Minimize Delete the large Group table then create a new smaller table 2 If this does not work from the View menu select the large group then choose Delete groupname from the Edit menu Save the file close the file reopen the file Checkbox for Plate blank This option is not visible until after data has been collected subtraction in the reduction dialog box is not visible Y axis units on a graph either This can occur when the range on the Y axis is very small Increase the Well Graph or Graph section height of the Y axis are repeated Ared hand appearsinthe A Visioneer PaperPort extension Macintosh is in conflict with Soft instrument information por Max Pro Disable or remove the PaperPort extension and restart the tion of a Plate section or computer CuvetteSet section Text appears too large for In the Windows Control Panel check the settings for Display Ensure wells in plate on Windows that you are using small fonts platform General display problems in Change the video display settings to 16 bit and high color or better Windows Printing Problems Text in the header field of the The text is still there but is not visible It will print If you click in the print dialog box disappears box and drag the mouse to the left it will reappear if you press the return or enter key The Notes section is longer Insert a linefeed int
283. display Kinetic and Spectrum modes only choosing Graph again returns to regular display toggle f Opens the Display dialog box containing Display in the CuvetteSet menu display Toce EE Display Mask in the CuvetteSet menu Masks the selected cuvettes ie l Includes excludes section from printed Printer icon report Section Name in the CuvetteSet Opens the Section dialog box allowing you menu to rename the section New Window in the CuvetteSet menu Opens the section in a new window DEN Copy Template in the CuvetteSet Copies the template allowing it to be pasted menu to another CuvetteSet section Paste Template in the CuvetteSet Pastes a previously copied template to a new menu CuvetteSet section Double click a single cuvette or several i selected cuvettes Graph in the Cuvette Set menu Opens the Export Template to dialog allowing you to export a template to an ASCII text file Export Template in the CuvetteSet menu Opens the Import Template to dialog allowing you to import a template from an ASCII text file Import Template in the CuvetteSet menu 3 18 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Group Section Tool Bar Body Summaries SoftMax Pro User s Manual ul TT Standards EA Standards pg ml Concentration Wells 590 000 590 000 Mean alue 530 000 510 000 610 000 560 000
284. dit Summary command 3 26 3 38 Emission scan spectral B 6 End wavelength nm 7 36 8 29 End Time 7 28 7 31 8 21 8 23 Endpoint 1 3 4 9 5 10 7 27 8 21 display defaults 7 6 8 6 reduction 7 27 8 21 Enlarged well graphs 7 18 8 14 Enterprise Administrator Pre Installation Information 6 3 Enterprise Edition 6 28 Error bars 7 53 8 51 Error messages 11 8 pasted text 11 8 E Statements 6 29 Excitation scan spectral B 6 Experiment active 3 9 3 30 menu 3 30 section 3 8 Exponential curve fit 7 63 8 60 B 6 Export command 3 26 9 16 data 9 16 format 2 17 Preferences setting 2 16 9 16 Export Enterprise Administrator 6 24 Export Options Enterprise Administrator 6 12 SoftMax Pro User s Manual File autosaving 9 9 creating 9 3 9 5 cross platform 9 7 data 2 9 2 10 9 5 management 9 3 9 5 menu 3 26 name number of characters allowed 9 5 9 9 opening 9 6 SoftMax 9 6 protocol 2 9 9 5 saving 9 8 10 21 10 53 saving manually 9 8 stationery 10 21 10 53 File protection 9 29 File sheilding 9 29 Filename extensions 2 10 filename extensions 6 2 Filter wavelengths 2 19 FLEX mode 1 4 5 40 B 6 Fluorescence 1 3 5 34 5 51 Fluorophore B 7 Format text 9 14 Formula 7 67 8 64 column 7 45 8 43 copy 9 27 custom 7 67 8 64 custom reduction 7 38 8 35 editing 7 43 8 40 help 1 9 plot 7 47 8 45 summary 7 46 8 44 Formula button 7 38 8 36 G G4 2 4 Global Options 6 22 Glossary of Terms B 1 Graph 7 47 8 4
285. divided into two parts left and right the default display Raw RFU or Raw RLU is shown to the left of a dividing line the choices for reduced data displays are shown to the right of the dividing line Note data reduction itself is performed separately settings here simply determine how the reduced data will be displayed in the Plate section The dialog boxes below show the default display choices for the three modes e Raw fluorescence or luminescence values numbers for Endpoint e The change in raw RFU RLU values over time for Kinetic e Raw RFU RLU values for the range of wavelengths displayed as a plot for Spectrum e Raw RFU RLU values as shades of gray for Well Scan e The change in raw RFU RLU values over time for FlexStation Other choices available in this dialog box include displaying the data as a sin gle reduced value number in threshold ranged or gray scale formats or any combination of display choices with the reduced number the reduced number display choice cannot be used with the reduced number checkbox SoftMax Pro User s Manual Endpoint Kinetic Spectrum Well Scan FLEX Figure 8 2 Plate Section Endpoint Kinetic Spectrum Well Scan and FlexStation Display Dialog Boxes Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments wm Display 2 Raw Reduced Raw RFU Example Raw RFU RLU default Show With reduced number Raw Data
286. double click the name of the Graph section Standard Curve to the right of the Graph section icon Export Graph Creates a file containing a picture of the graph with a filename of your choice On the Macintosh the graphic is in PICT format in Windows the graphic is in EMF Enhanced MetaFile format New Window Opens the active section in a separate window Mouse Equivalent double click the 3 icon in the Graph section SoftMax Pro User s Manual 3 37 Chapter 3 SoftMax Pro Overview 1 F 3 36 Group Menu Create Column Create Summary Edit Column Edit Summary ewer Set tine JT 1 d x Y pal Lf h fi Se f ee n Hide H Hide Replicates Show All Show Formulas Group Settings New Window This menu item changes to show CuvetteSet Plate Notes Graph or Group depending on the type of section that is currently active If no section is active this menu item does not appear Create Column lIf a Group section is currently active you can add a column to the display by choosing this command If no columns are highlight ed the column will be added at the end of the group If you select a column first the new column will be added to the right of the high lighted column Button Equivalent UU in the Group section Create Summary Allows you to create a summary and enter a formula for this summary for the Group Summaries appear at the bottom of the group table Button Equivalent
287. e line using linear regression after the wavelength combination reduction This setting uses all visible timepoints in the reduction window Slope is the same as Vmax Rate when Vmax Rate is set to the same number of points as the run i e the default Slope is not the same as Vmax Rate if you have modified the Vmax Points Area Under Curve This reduction estimates the area under the curve as defined by the data plots in wells and cuvettes within the reduction limits The data plots are treated as a series of trapezoids with vertices at successive data points and at the X axis coordinates of the data points The areas defined by each of the trapezoids are then computed and summed SoftMax Pro User s Manual 7 35 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 7 36 Spectrum SpectraMax Instruments Only Spectrum Limits The Limits settings for the display of Spectrum data are MaxOD MinOD Starting Wavelength in nm and Ending Wavelength in nm The display of values is shown relative to the first point measured for each well Limits define the data that will be viewed and included in data reduction but do not affect data collection If you alter a limit to show less data you can always display the hidden data again by changing the limit MinOD The limit for the minimum value you wish to report Any values from the reading that are under this limit will not be shown and will be excluded fro
288. e Figure 2 1 below to connect them to a Macintosh having a serial port This cable is NOT the same as a Macintosh printer cable Connect one end of the cable to the Macintosh to either the printer or modem port and the other connector to the MDC Microplate System instrument Use an 8 pin DIN to DB 25 Macintosh modem cable to connect the Macintosh to an Emax Vmax UVmax or ThermoMax Connect the 8 pin round DIN connector to the Macintosh to either the printer or modem port and the 25 pin connector to the MDC Microplate System instrument 2 2 SoftMax Pro User s Manual Chapter 2 Installation peut ttt lc AAA Figure 2 1 Serial Cables for Macintosh Left 8 Pin DIN to 8 Pin DIN Serial Cable not the same as a Macintosh printer cable Right 8 Pin DIN to DB 25 Modem Serial Cable Connecting the 8 pin end of either cable to the modem port of the Macintosh is preferred since this allows the printer port to remain available If you do con nect to the Macintosh printer port first deactivate AppleTalk using the Apple Talk Control Panel or the Chooser desk accessory in the Apple menu If you are running other applications that use AppleTalk it may be necessary to quit them before making AppleTalk inactive USB Connection iMac and G4 Macintosh computers do not have a serial port You can connect a serial cable to these Macintosh models and the MDC Microplate System instrument through the use of a USB to serial adapter
289. e Plate sec tion The instrument will use these settings to read the plate Table 5 1 Features Available in the Instrument Settings Dialog Box for Fluorescence Instruments Wavelength Sensitivity Readings Read PMT Well Bottom Compound Source Transfer AutoCalibrate Assay Plate Type Type Timing Sensitivit Cutoffs Scan Read Pipette Tips Layout Compound Wells to Read AutoRead yP y Editor amp Tip Column SpectraMax Gemini Fluorescence Readers Endpoint Fluorescence Luminescence Time Resolved Fluorescence Kinetic Fluorescence Luminescence Time Resolved Fluorescence Spectrum Fluorescence Luminescence Time Resolved Fluorescence Well Scan Fluorescence Luminescence Time Resolved Fluorescence FlexStation Endpoint Fluorescence Luminescence Time Resolved Fluorescence Kinetic Fluorescence Luminescence Time Resolved Fluorescence Spectrum Fluorescence Luminescence Time Resolved Fluorescence Well Scan Fluorescence Luminescence Time Resolved Fluorescence FLEX Fluorescence No Automatic setting No Automatic setting default is no cutoff filter Default is no cutoff filter The SpectraMax Gemini EM can perform bottom reads but does not support Time Resolved Fluorescence mode 5 Fluorescence is the only read mode available with the FlexStation no Reader choice available no top read choice is available in FLEX mode Nose Incubator control is an instrument setup p
290. e Plot X dialog and drag to select one of the choices in the menu To disable error bars choose No Error for the X and or Y axis Error bars show both positive and negative error Depending on the range set tings for the graph portions of the error bars may not be visible Curve Fitting When you first create a graph of the data it does not have a fit associated with it The graph might appear something like this v 7 Graph Fit Graph No Fit selected 60 concentration Plot 1 Group 1 concentration vs Mean value Figure 8 39 Graph with No Fit SoftMax Pro User s Manual 8 51 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 1 k 8 52 You can fit any plot to one of nine curve fitting algorithms Linear Semi Log Log Log Quadratic 4 Parameter logistic Log Logit Point to Point Expo nential and Cubic Spline These selections are shown in the Fits pop up menu in the Graph section toolbar click on the box next to Fit Typically a standard curve refers to the curve fitted to the plot of concentra tion versus mean value for the Standard group Once you have selected a Fit type SoftMax Pro determines the parameter val ues that best fit the data The function with these parameters is then drawn on the graph Ideally the type of fit used should be determined by the underly ing chemistry of the assay and could be set before data is read A g
291. e and the following directory and file string XXX Example Without the attribute ExportAs c SoftMax Pro exportfile txt With the attribute ExportAs a c SoftMax Pro exportfile txt Importlemplate X XX Have SoftMax Pro import a template file The file should comply with Soft Max Pro s template file format A plate section should be selected prior to importing a template NameCurSection New This re names the current selected section If no section is selected nothing happens Notes e This command is used typically to identify different plates in an automated environment e The data copied will follow the display settings for the current plate nor mally Raw Data Example NameCurSection Flatez 0025345 Have SoftMax Pro send a New command This creates a new document and reads in the values contained in the Default Protocol ppr protocol file NewNotes Tell SoftMax Pro to send a New Notes section command This creates a new Notes section within the current Experiment NewPlate Tell SoftMax Pro to send a New Plate section command This creates a new Plate section within the current Experiment OpenAssay X XX Have SoftMax Pro open a protocol file The file should be in SoftMax Pro s Assays folder which is set using the Assays menu Protocols can also be stored in subdirectories of the Assays folder Notes e Only protocol files can be opened e SoftMax Pro will append ppr to any filenam
292. e destination Plate section and choose Paste Template from the Plate menu Template Reduction Template Reduction Display Graph Mask Copy Template Paste Template Display Graph Copy Template Paste Template Export Template Import Template Export Template Import Template Section Name New Window Section Name New Window Figure 5 32 Copy Template and Paste Template Commands in the Plate Menu The group names and sample names of the pasted template are identical to those of the source template In effect the destination template is considered an extension of the original template and all wells on the destination plate are considered replicates of the wells on the source plate same group and sample names Any changes made to the template on the destination plate will also be made on the source plate Data from the two Plate sections will be analyzed together Copying and Pasting between Different Experiment Sections SoftMax Pro User s Manual To copy a template make the section containing that template active and choose Copy Template from the Plate menu the menu name changes to reflect the type of active section Then activate the destination Plate section and choose Paste Template from the Plate menu The group names and sample names of the pasted template will be identical to those of the source template but since they are in different experiments the full
293. e eT te UIS AELE E AE E E EN RH RUE ARIE AN E S E E ELL E VMAS ENA RN hi B sz i m L m Well Scan Reduction M a rimum Limits mn H a ILm1 w Average Min RFU TIE Max RFU pesseu eh ehh eh eh ERR RR ERR NER RRE RR RRE ERR RRR ERR RRR NER ER EER ERR RR ERE RRR RR ERR ER RR E ER EER Iu Iu su su au eau sh ehh nnn nnn nnn nnn nnn nn nnne nnn wv Vilas units per sec Time to VMax xi Onset Time B Wavelength CORT ME REN srr a f N desi IET See ee eee ee eee eee ee Time at Maximum Mer LIE ee Passaes0PeoPe0PP00P009P00000000000P090P000P009000000000000000000000090000000000000000000000000000009 900000000000 00000000000 90000000000000000 000000000000 00 0000000000000 09005 Time at ue Kam E LAE eoe ie Bassihe Obllone 9m T Lm 1 Peak i MnRFU o tesTme op Absolute Max Min Max RFU End Time C Zero Baseline Points a Oo Mutipler hd Moving Average 3 Points E Interpolate Raw Data Mean Cancel Custom Figure 8 13 Reduction Dialog Boxes for All Modes 8 20 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Endpoint All of the read modes offer one or more predefined choices for reduction The predefined reductions are described below under the appropriate read mode heading In addition with any read mode you can cre
294. e eee eee 9 24 New vs Duplicate within an Experiment 0 0 c eee eee eee eee eee 9 28 Actions Protected by Password 224454 i448eesse re PRI WRE DE RUPEE pd ces 9 30 Instrument Settings for Absorbance and Fluorescence Instruments 10 9 Instrument Settings for FLEX Mode sssseeeeeee eens 10 39 SoftMax Pro User s Manual xxi t xxii SoftMax Pro User s Manual Chapter 1 Welcome to SoftMax Pro TatroCcLe LO Dude nett m greet erases dead Y su de aen ute LC otv ete ae 1 3 New to SoLt Max PIO boss ret pe ar Me PR E Rus 1 4 What s New in SoftMax Pro Version 4 3 0 0 cece ee ee eee 1 4 About the Documentation 0 0 00 ccc eee eee eee eee nas 1 6 Conventions Used in This Manual 00004 1 7 Getin Rel Die uie Ae cuv du CUR soe anal ap ua ae yom ohne Sais ad ag 1 9 Macintosh On Line Help 2 cece eee e eee 1 9 Windows On Line Help uan c2cenetaecehowcer tere seers 1 10 Troubleshooting and Technical Support 1 12 Chapter 1 Welcome to SoftMax Pro t 1 2 SoftMax Pro User s Manual Chapter 1 Welcome to SoftMax Pro Introduction SoftMax Pro User s Manual SoftMax Pro is designed for use with Molecular Devices Microplate Readers Emax Vmax UVmax ThermoMax and VersaMax Micro plate Spectrophotometers SpectraMax and Microplate Spectrofluorome ters SpectraMax Gemini fluorescence reader family and FlexStation i
295. e in signal called the onset OD or RFU or RLU to occur Open Close Triangle A small triangle located on the left hand side of section and experiment tool bars Used to open close sections and experiments within a SoftMax Pro file Optical Density OD The amount of light passing through a sample to a detector relative to the total amount of light available Optical Density includes absorbance of the sample plus light scatter from turbidity Option Button Radio Button A small round button that appears in a dialog box or window Within a group of related option buttons you can select only one Es Parallel Port Windows 2 A connection on a computer usually LPT1 to plug in the cable for a parallel printer Windows supports parallel ports LPT1 through LPT3 B 10 SoftMax Pro User s Manual Appendix B Glossary of Terms Paste A function that copies the contents of the clipboard and inserts this copied information at the current selection or cursor position The paste function is enabled by pressing 36V on the Macintosh and CTRL V in Windows Path Windows Specifies the location of a file within the directory tree For example to specify the path of a file named ASSAY BAT located in the SOFTMAX directory on drive C you would type c softmax asgay bat PathCheck Instrument setup option within SoftMax Pro for the SpectraMax Plus Plus 190 or 340PC that allows data collected in a Plate section to be normalized to a
296. e individual wells for saturation If the saturation occurs at the time or wavelength of interest you will need to reread at a lower PMT setting If the saturation does not occur in the time segment or wavelength of interest you may ignore the message and proceed with data analysis Chapter 11 Troubleshooting Error Message Boxes Error messages contain a stop graphic O or e and text explaining the error if the software can report it Some errors such as an obstruction of the drawer of the microplate reader report the problem allow you to correct it and then proceed with the assay Other errors are not recoverable and require that you perform the action or assay again Error messages are listed below in alphabetical order by first line in the mes sage Instrument reported a fatal error SUM Tmax Pro Instrument reported a fatal eror Please contact customer support This error appears when the instrument experiences a hardware failure or if a jam occurs in the tip box for example The run in progress is not recover able Call Molecular Devices Technical Support to receive information that is pertinent to the particular situation that led to receiving this error message Pasted text doesn t match plate settings O Pasted Text doesn t match plate settings You have attempted to paste values from an external file containing ASCII tab delimited data into a Plate section or CuvetteSet section but the numb
297. e limited to 31 characters on the PC the limit is 59 characters for the actual name of the file plus a period and 3 letter extension for a total of 63 characters Using a PC it is possible to make a file name longer than this by man ually changing it in a directory listing or by using the Save As com mand in the software A file with a name longer than this limit will no longer be able to be read by SoftMax Pro and will return an error when you attempt to open it File names that are made too long using the Save As command will show corrupted characters such as y etc for any letters that fall after the 63rd SoftMax Pro can be configured to save a copy of your data file automatically Autosave after a reading This function is enabled in the Preferences dialog box choose Preferences from the Edit menu Preferences Serial Part Serial Comm Speed 9600 w penne 7 y Autosave Si m Export Format x j 1 SOFTma PAO 4 0 9 Time Plate LJ Append Date bd Include Labels SHE Frem C Append Time O Time Interpolate Add Filename and Date Pere TEC ET ee Cee ee Eee eee MCLEE ee ee EEE ECE EME CET Cee CEre ree Te rE eer eC eer errr amp SOFTmax PRO File 3 Text File RRNSSRESARRRRRRRRRARRSARSRRRRARRRARERRERARRRRARRARRANRRNRRERRERNSRRRRRRRERANRRRRRRARSNARRARMRARRNAREARRRARRIARSRRNRARRERRRAREREEEANARRNRARRREIRERRSNARRRREESRENARRR RRE RRRARR RA EE Rea
298. e microplate chamber This is differ ent from the front panel of the instrument which displays the temper ature within the cuvette chamber The readings should be very similar to one another after both chambers have reached equilibrium During warm up however you may notice a discrepancy in temper atures which is normal SoftMax Pro User s Manual 3 7 Chapter 3 SoftMax Pro Overview 1 F The Read button If a single Plate or CuvetteSet section has been created in the experiment window clicking the button starts the reading This reading is based on the settings chosen in the Instrument Settings dialog box refer to Instru ment Setup for the plate If more than one Plate or CuvetteSet section has been created in the experiment window and no Plate or CuvetteSet section is currently active clicking the button opens a dialog box asking you to select which plate or cuvette to read If a CuvetteSet or Plate section is active when you click the Read button that sec tion is read automatically The button If a CuvetteSet section has been created and is active this but ton will be displayed Clicking the button will reference the cuvette The ref erence is based on information entered in the Instrument Settings dialog box The reference reading may be taken before or after the sample reading This setting ap plies only to the SpectraMax Plus and Plus 9 The Incubator button With ThermoMax VersaMax SpectraMax
299. e passed in SoftMax Pro User s Manual Chapter 12 SoftMax Pro Remote Command Language e If the file is not found SoftMax Pro does nothing Examples OpenAssay myFile OpenAssay mySubDirectory myFile OpenCDrawer Flex Station only Tell SoftMax Pro to open the compound drawer OpenDrawer Tell SoftMax Pro to open the instrument drawer OpenTDrawer FlexStation only Tell SoftMax Pro to open the tips drawer ReturnData This command asks SoftMax Pro to send data back from the currently selected plate Generally this is used to send the data directly to the caller instead of using the clipboard If data is to be returned to the clipboard this command functions like the SoftMax Pro Copy command when a plate is the currently selected section ReturnStatus This command returns the current instrument status Even if queue operation is enabled this command is not queued and is processed immediately The status is an ASCII string that has e Information about the instrument type and version e Whether the instrument is busy or idle e The instrument s current temperature and e The state of its drawer If queue operation is enabled the number of remote commands queued is also returned The exact format of this response depends on the type of instrument in use If SoftMax Pro failed to place data in the clipboard then no return status will be available SoftMax Pro User s Manual 12 11
300. e plate or enter a different value from 1 to 999 seconds in the box to the right Between Reads To shake the microplate between readings click the checkbox next to this set ting For readings at a single wavelength Between Reads will shake the plate for the set amount of time prior to each reading at that wavelength For readings at multiple wavelengths enabling Between Reads will shake the plate for the set amount of time before each reading at the first wavelength only You can accept the value shown for the amount of time to shake the plate or enter a different value from 1 to 999 seconds in the box to the right Compound Source This allows you to select from various types of plates used to store fluid com pounds that can be withdrawn and then injected into plate wells during the run The type of plate that is chosen for this setting should match the actual plate used in the Compound Source drawer and the number of wells selected in the Assay Plate Type Compound Source Select a compound plate type Beckman 140504 Costar 3957 Costar 3959 Costar 3961 Costar LIbtm Greiner btm Polytitranics 7701 5400 Figure 5 44 Compound Source Settings Box SoftMax Pro User s Manual 5 45 Chapter 5 Reading Using Fluorescence or Luminescence Instruments t 5 46 Compound Transfer Compound Transfer S Assay Plate Fluid Initial Volurne E Transfers S 3 y y Transfer 1 Tra
301. e plot name and clicking the button Clicking the New button opens the Plot X dialog box allowing you to cre ate a new plot for the graph Existing plots can be edited by highlighting the plot name and clicking the button which opens the Plot X dialog box containing all the information for the selected plot When creating a new Graph section the Graph Options dialog box opens after choosing either Or in the Plot X dialog The new Graph section is not created however until you click in the Graph Options dialog box SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Graph Options Name of the plot Fonts options you can change this Beni neinte OP ype onts lt Chart options box MO Scatter sin Sample Text i Itie Buc r1 Cluster bar s Sample Text hil Stack bar Legend Delete plot button dA enses bU Plot symbols New plot button X and Y axis Pew J Baie c labels Edit plot button i p Plot 5 Concentration Standards MeanaluedmeStandard Plot name and symbol a X and Y error bar tes ee description Figure 7 35 Graph Options Dialog Box Creating Multiple Plots SoftMax Pro makes it possible to create multiple plots within a graph Each plot will represent data from a group that has been created in the Template Editor or data from a summary or custom formula You may plot data from any group or from several g
302. e plots within a single graph after which is an example that provides more detail Multiple Plots Step Summary Create all required groups in the Template Editor e Read the microplate to obtain data e Create a graph section e Add plots to the graph and choose the group and the X and Y values to be shown for each plot Example of Creating Multiple Plots This example shows how three groups all of which are Standards were plot ted on the same graph 1 Groups are created in the Template Editor For this example four groups are cre ated three for standards Standard1 Standard2 and Standard3 and one for Unknowns Unknownl SoftMax Pro User s Manual 8 47 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Experiments 1 Plate 1 Broun ais Sample Unko1 Jw Series f Assign Clear 10 11 12 Cancer 9k Figure 8 33 Standards and Unknowns Groups Created 2 The associated Group sections are created using the Standards column format which by default generates columns for concentration and optical density val ues The example below shows the Group section for Standard2 kd Standard ry E r Standard pg ml Sample Concentration Wells Values _Meanvalis StdDev CV ETT RN tig TT E 420 000 420 000 0 000 Stal 0 500 410 000 410 000 0 000 00 00 Stao 0250 3 ooog 410 000 0 000 0 0 Stad 0125 C4 10 009 410 000
303. e reading but will use a simulator program built into SoftMax Pro to generate data that you will manipulate as if it had been obtained from an actual reading The same data is also contained in a file provided with the original software from Molecular Devices called Endtut pda The data in this file was generated using the parameters you have just created in this protocol file Step 7 Read the Plate If you were reading an actual microplate at this time you would prepare the microplate as it is defined in the Template Editor Before an actual reading you might find it helpful to print a copy of the template to assist in properly filling the plate You would then place the filled microplate in the drawer of the MDC Microplate System instrument Since we are using a simulator you don t need to load anything in the instrument To enable the simulator hold down the Ctrl and Shift keys and then choose Enable Simulator from the extended menu choices that appear in the Control menu see the figure below Control Instrument Setup Read Er 3 R Open Drawer Incubator eibi eir Enable Simulator Glear Errar Log Figure 10 22 Extended Choices in the Control Menu Now that the simulator is active click the button in the SoftMax Pro status bar at the top of the screen to initiate the simulated reading Values will appear in the data display in the Plate section as if they were being read by the MDC Microplate System in
304. e samples must be arranged sequentially in either ascending or descending order To create a series do the following 1 In the Template Editor highlight the wells to be part of the series The wells must be in the shape of a rectangle Experimentti Platest Grup Stdade F E Seer m Concentration t Series Group Name Button Pull Down Menu Sample Name Box and Selected Wells Units Figure 5 27 Template with Wells Selected for a Series 2 Click the Series button to open the Series dialog box Series Start From First Sample Sta0l s iw Top EJ Sample Descriptor B OBottom memean vts par e E5 O Left Starting value Feplicates 5 Cancel Figure 5 28 Series Dialog Box 3 Specify the name of the first sample in the series in the box or accept the default provided by SoftMax Pro Subsequent replicates use this name as a base and either increment the number within the name or append a number to it In the example above the sample name provided by Soft Max Pro describes the first sample 01 in a group named Standard here shortened to Sta 5 26 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual 4 5 6 The information shown in the box labeled Sample Descriptor is derived from the information entered in the Group Settings dialog box If the in formation for sample descriptor or units
305. e section s Print 7 CTRL P With the Macintosh choose this command and then click either Print all or Print report Print all sends all sections to the printer in the order they appear in the window Print report sends those sections selected to be in the report to the printer in the order they appear in the window From Windows choose Print and then specify a page range or leave the default to print all pages Make sure you have selected the correct printer prior to choosing one of the Print options in the File menu If you start to print and dis cover the wrong printer is selected choose Cancel to exit from the Print dialog box select the correct printer using the Chooser or the Print Setup command and then repeat the printing process Quit 6Q CTRL Q Closes SoftMax Pro as well as any SoftMax Pro files you may have open If you have open files that have changed and have not yet been saved a dialog box will ask if you want to save them before quitting 3 27 Chapter 3 SoftMax Pro Overview Edit Menu Undo 3 7 CTRL Z Restores the last text operation to the condition that exist Unda LUT Copy a6 Paste EY clear Select All Delete Plate 1 Duplicate Platezx1 art Of sl t th LIED ft L3 Ly C oon Preferences Suspend Recalculation Recalculate Now 3 28 ed before the editing was done used primarily when entering text in Notes s
306. e that the tip of the pipette does not en gage the fluid in the well to avoid cross contamination and to allow re use of tips Note that subsequent transfers will require that you calculate the amount of fluid added and set the pipette height accordingly higher to avoid touching the fluid in the well e Rate from 1 to 8 This setting determines the rate at which the fluid is pipetted into the well of the microplate A setting of 1 is equal to 26 microliters second and each subsequent number increases in increments of 26 microliters Therefore a setting of 2 is equal to 52 microliters etc A setting of 3 to 4 may help to minimize cell damage or splashing SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments e Volume from 1 to 200 uL This setting determines the volume of material that will be pipetted from the source to each individual well chosen to receive that transfer e Time Point from 5 to 9999 seconds This setting determines the time after the start of the run when the fluid is scheduled to be dispensed Minimum time This information line lists the minimum time required before a pipetting event can occur The Time Point setting above cannot be set to a number of seconds smaller than this value This minimum time value is cumulative not an interval between pipetting events The value is the minimum number of seconds of elapsed time from the beginning of the read It takes into a
307. e upper right are not a separate group the samples also named BL are contained within the boundaries of the large group called Unknowns and are the same color as that group In order to save changes you have made to the template in the Win dows version of the Template Editor be sure to click the button to close the window rather than clicking the Control Menu box Click ing the Control Menu box located in the upper left of the window performs the same function as the button the window will close but any changes you have made to the template will not be saved SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Copying and Pasting Templates Copying Complete Template Information Two template actions Copy and Paste can only be activated from the Plate menu when a Plate section is active rather than from the Template Editor If you have created a template in one Plate section and wish to use it in another Plate section you may copy that template and paste it into the new section The results of copying and pasting a template depend upon whether the action is performed within the same experiment or between experiment sec tions Copying and Pasting within the Same Experiment Section To copy a template make the section containing that template active and choose Copy Template from the Plate menu the menu name changes to reflect the type of active section Then activate th
308. e will only apply after the wavelength combination Baseline with Multiplier This option has been added to make FLEX data look more like FLIPR data FLIPR data can be imported into SoftMax Pro and this option modifies the plot and reduction calculations to make FLEX data comparable to the FLIPR data This method averages the first number of points you specify the upper box divides the raw data by this average value and then multiplies by 100 The Multiplier section allows you to enter a number from 0 0001 to 9999 which is then used to multiply the Baseline value This calculation also is performed only on the reduced data and only after any wavelength combinations are calculated To see the modified plot you must choose the Reduced Plot button in the Display dialog box or click the Show Reduced button in a zoomed well graph window Interpolate Raw Data Checkbox E interpolate Rawe Data Figure 8 21 FLEX Mode Reduction Dialog Box Interpolate Raw Data Checkbox Each individual raw data point has a corresponding read time associated with it In FLEX mode each data value is logged with its own individual read time which can make it difficult to perform wavelength combination calcula tions correctly For example if you desired to take a ratio of two different SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments wavelengths in a si
309. e4 Sed Figure 10 66 Plate Display with Data Displayed as Gray Scale 10 56 SoftMax Pro User s Manual Chapter 10 Tutorials Step 8 Data Analysis Group Sections A SoftMax Pro User s Manual Group sections are created automatically by SoftMax Pro when you define groups in the Template Editor In this tutorial new group sections were added when you created the new groups Data and Control The content of Group sections changes depending on the wells that are assigned to these groups in the Template Editor Before a plate is read these sections show no data for the wells After a reading Group sections contain information about the values for the wells contained within the groups Scroll down and click the small triangle on the tool bar of the Data Group it opens and is active It should look similar to this e Ea i r Data Datan1 Ad 0 000 dsaon az200 000 100 000 2 3 B1 458200 0 C1 4000 0 D1 476000 E1 47 200 0 F1 46700 0 G4 468100 0 H1 45400 0 Data Ad 0 010 48200 46975 000 09522 25 B2 458100 0 cP 47200 0 p2 47400 0 E 47000 0 F2 464000 G2 45800 0 H2 46000 0 Dataos amp 3 0 050 448200 0 46575 000 0676 29 Ad 48400 0 B3 428000 0 B4 477800 0 c3 47 600 0 c4 474000 D3 47 200 0 D4 47000 0 E3 46700 0 E4 456400 0 F3 486100 0 F4 455800 0 63 45400 0
310. eSet section Each template is built upon a hierarchy as follows the template is composed of samples and groups A set of one or more replicate wells makes up a sam ple A set of related samples forms a group For example you may have a group named Standards that consists of seven samples named STD01 STD02 STD07 Group names may be up to 32 characters long while sample names may be 300 characters long Samples may be assigned one at a time using the Assign button or several at the same time using the Series button If you create a group in the Template Editor but do not assign any wells to the group a section for that group will still be created automatically when you close the template You can remove the group both its section in the window and its group name in the template by making that Group section active and choosing Delete Groupname where Groupname is the name assigned to that group in the template from the Edit menu Using the Macintosh you can also delete the selected Group by pressing within Windows you can press CTRL4 Note that deleting the wells assigned to a group from the tem plate does not delete the corresponding Group name from the drop down list The only way to remove a group name from this list in the Template Editor is to delete the corresponding Group section from the experiment 4 21 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 1 k 4 22 Selecting Well
311. ea that shows the experiment name with the mouse Selecting an experiment causes its tool bar to change from light gray to darker gray and the experiment title to be shown in boldface type Selecting is different from activating an experiment Selecting an experiment allows you to perform actions on the entire experiment such SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview as deleting or duplicating it Multiple experiments can be selected by holding down the Shift key while clicking the individual tool bars of the experiments A single click anywhere within an inactive experiment activates it Activating an experiment allows you to add new sections to it or to perform other opera tions within that experiment With sections however selecting and activating are identical functions Clicking anywhere within an inactive section activates and selects it You can create a new experiment by choosing New from the Edit menu Choosing Duplicate from the Edit menu creates an identical copy of the cur rently selected experiment excluding data The new experiment retains the same name as the original with the word Copy appended when you choose to Duplicate e g the duplication of Experiment1 produces Experimentl Copy and an increment of number when you choose New e g Experiment1 becomes Experiment2 An experiment can be deleted if it is no longer needed The Delete command in the Edit menu changes depe
312. ead Entire Plate Strips Selection SpectraMax and VersaMax Instruments Only You may choose to read only certain columns or strips of wells in the micro plate or to read the entire plate Partial plate reading is available for all read ing modes although it is most useful for fast Kinetics and limited Spectrum analyses The time required for Kinetic and Spectrum readings may be signif icantly reduced using this setting since the instrument does not have to read the entire plate To enable a partial plate read highlight the desired columns see Figure 4 15 Columns do not need to start with 1 but must be contiguous PW ue X Wr Xr Ar K 4 d FPE LR RR CR LR SUM M eu RM ox Korn wr CC XC COL T COICO F C s E e Je Q ll M M n be ban Be mm wem o KU c7 Xm ic Ir mmo om 0 M O C C C C Q E OOGOQCGOCOCOC N COCOCOC OC ees OOGOQOCGOCOCCP OOGOGQOCGQXCXC XC OOGOGQOGCGQCXCXC J OOGOGQOCGOCOCCP COCOCOC OC COL C d Figure 4 15 Partial Plate Read Strips Dialog Box with Eight Strips Selected If you enable a partial plate reading only the columns to be read will be visi ble in the data display indicating that no data will be collected for the other wells all columns will be present in the Template Editor however AutoCalibrate This checkbox allows you to disable or enable automatic calibration for all instruments T
313. eae eee Lm3 Log10 Lm1 Lmn Average lallvalues This formula will average together the relative fluorescence lumines cence units for multiple readings at the same wavelength for exam ple if you read the well six times at a single wavelength 6 36 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Table 8 3 Wavelength Reduction Formula Examples oon o o0 oo0me Min allvalues This formula will report the minimum RFU RLU recorded for multiple wavelength readings in each well Max allvalues This formula will report the maximum RFU RLU recorded for multiple wavelength readings in each well If Lmz A makeerr 118 if L This formula will report low for any well with an RFU RLU less mx gt B makeerr 117 Lm1 than A high for any RFU RLU greater than B and the RFU RLU of any well that lies between A and B Custom reduction formulas utilizing mathematical operators or terms can be used to obtain specific types of data Table 8 4 provides some examples of such formulas for Kinetic and Spectrum readings Table 8 4 Kinetic and Spectrum Reduction Formula Examples Vmaxcorr combinedplot Reports Vmax correlation coefficient for plots in all 96 wells Ivmaxpoints readinterval Vmax Delta combinedplot Will report the Vmax Rate of the delta between each time point Ivmaxpoints readinterval Spectrum or Kinetic Nthitem Lm1 X Will report
314. earance of the Reduction dialog box will be dif ferent depending on the type of read mode you have chosen and the options you have chosen in the Instrument Settings dialog box Figure 8 13 shows the Reduction dialog boxes for a Plate section in Endpoint Kinetic Spectrum Well Scan and FLEX modes along with the default reductions for each of those modes 8 19 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Reduction Dialog Boxes Default Reductions Reduction Custom Endpoint PR M Use plate blank V max v V max Cunits per sec Time to Ymax Onset Time Custom Time at Minimum Kinetic a Ibert Time at Maximum Time at 1 2 Maximum Slope Area Under Curve Wavelength Combination 1 y Use plate blank Lag Time p j Absolute Values End Time Cx Maximum Minimum x Lambda at Maximum Spectrum Custom Lambda at lTinimum x ILral Area Under Curve ms Reduction X d Wavelength Combination dg unuc csevespeecsescesseessscansesecsousvesceusenecceessccsnsesessuennssnecesesd sncesssusns soos cnccsocsssbesessoeesecsssunscssoueansesssessdusccscsceeceeesessesses IR aoe rer
315. ece of text in that column Double clicking the line dividing two columns causes the single column to the left of the divider to be sized according to the largest amount of text in that column Showing and Editing Existing Column Formulas When group sections are first created the formulas used to calculate the data are not displayed These formulas can be shown until you choose to hide them again by mak ing a Group section active and then choosing Show Formulas from the Group menu Note that the command in the menu changes to Hide Formulas after they are shown Create Column Create Summary Edit Column Edit Summary AWULOSIFe Hide H Hide Replicates show All Show Formulas Show Formulas Command Group Settings New Window Figure 7 30 The Show Formulas Command in the Group Menu Choosing this option causes an additional row to appear beneath the head ing for each column which shows the formula used to calculate the data in each column To see formulas briefly hold down the shift and control Ctrl keys SoftMax Pro User s Manual 7 43 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments iii Standards l 24 24 Standards mg ml Concentration Wells values MeanV alue Std Dev CY ISample Concentration lwellll weTlvali Averagel Yalu StdevivaTu Cv val 0 000 B1 D 252 0104 41 2 Formulas Smallest standard value FTin lTean Value 0 253 Largest stand
316. ecked Click to close the dialog box SoftMax Pro User s Manual 10 19 Chapter 10 Tutorials t 10 20 Raw OD Absorbance ae Step 5 Set the Display Parameters Click the Display button to open the Display dialog box ww Display E 2 Raw Reduced m Display 3x Cres AOR BZ ONE NN EUER Paw Eaduead 2 Raw RFU 0330 Example RRRERREERRESREESEESESEESEREESENSREREEERRRERAER Show With reduced number with reduced number Raw Data in 1000 s Fluorescence Cancel Figure 10 19 Display Dialog Box The default display for Endpoint readings is raw OD for absorbance instru ments and raw RFU for fluorescence instruments It is not necessary for the purposes of this tutorial to show a reduced number so make sure the check boxes next to the options under Show are not checked If you decided later that you would like to view the reduced data you can change the display options after the reading since this does not affect the actual data only how it appears on the screen and printout Click to close the dialog box SoftMax Pro User s Manual Chapter 10 Tutorials Step 6 Save the File SoftMax Pro User s Manual Choose Save from the File menu Because the file has not been saved before and is not named the Save As dialog box will open Tutorial Format 5oftMAX PRO Document 5 0ft MAX PRO Protocol Figure 10 20 Save As Di
317. ection Showing 50 Location on Plot A summary formula is created by either clicking the summary button in the tool bar or choosing Create Summary from the Group menu A Calculation dialog box opens allowing you to enter the formula for the summary and to set the options for how the summary is displayed The same Calculation dialog box appears when you edit an existing formula Double click the summary at the bottom of the Data section just above the Graph section The Calculation dialog box that appears should look like the one below Calculation Name Summarys I L Hide Name Description EC 50 Formula InterpX Plot 1ivGraph 1 50 Decimal Places 5 Accept Figure 10 81 Calculation Dialog Box for EC 50 Summary Formula If you wanted to interpolate a value at a different percentage you could enter a different number at the end of the formula in place of the 50 there You could also reverse the formula to show the percent activation that is interpo lated for a certain concentration 0 1 in this example by writing the for mula this way InterpY Plot 1 Graph 1 0 1 The result would be 69 494 to three decimal places The summary can be shown with different accuracy up to nine decimal places if desired by changing the number in the box next to Decimal Places at the bottom of the box 10 66 SoftMax Pro User s Manual Chapter 10 Tutorials Step 11 Print a Report SoftMax Pro Us
318. ection dialog StandardCurve tion box double click Graph Settings in the Opens the Graph Settings Graph menu dialog box Drop down menu allows you to choose a curve fit X Axis in the Graph Opens the X Axis Settings menu dialog box Y Axis in the Graph Opens the Y Axis Settings menu dialog box l Includes excludes section Graph Options in the Opens the Graph Options Graph menu dialog box Export Graph in the Opens the Export Graph Graph menu As dialog box 3 22 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Instrument Settings You must specify the type of reading to be performed prior to actually read ing a microplate or a cuvette Instrument settings allow you to choose a read mode Endpoint Kinetic Spectrum Well Scan or FLEX a read type fluorescence time resolved fluo rescence or luminescence if using an instrument that performs fluorescent readings the number of wavelengths or the range of Spectrum data timing for Kinetic runs and so on Which settings are available depend upon the type of instrument connected or chosen in the Preferences and whether you are reading a plate or a cuvette Complete information regarding instrument settings can be found in Chap ters 4 or 5 choose the chapter that is appropriate for the instrument and or read mode you are using Menus and Commands bl The menu bar for SoftMax Pro is located at the top of the window just beneath the title
319. ection or CuvetteSet section tool bar Read In a single plate experiment the Read command reads the plate or cu vette immediately If no Plate section or CuvetteSet section is active in an experiment containing multiple Plate sections or CuvetteSet sec tions the Read command displays a dialog box asking you to select which Plate section or CuvetteSet section to read The default Plate section or CuvetteSet section selected by pressing the Return key is chosen on these criteria e The selected Plate or CuvetteSet section if one is selected e The next unread Plate or CuvetteSet section e The first plate or cuvette if all have been read Once a reading is in progress menu items are no longer available until the reading is finished Button Equivalent in the Status bar Ref Available only when a CuvetteSet section is active and a SpectraMax Plus or Plus is connected or chosen in the Preferences This command causes the SpectraMax Plus or Plus to read the cuvette in the cuvette port and applies the reading as a reference to all cuvettes in the Cuvet teSet section Button Equivalent in the Status bar Close Drawer Closes the drawer at which time the menu item changes to Open Drawer The drawer opens or closes immediately Button Equivalent E H in the Status bar Incubator Available with ThermoMax VersaMax SpectraMax and FlexS tation instruments only Allows you to turn the incubator on or off
320. ections This menu item is dimmed if no actions have been taken or if undoing the last action is not possible Cut 36X CTRL X Removes highlighted information text or data from the document and stores it on the clipboard so it can be pasted elsewhere This menu item is dimmed if no selection has been made or if the se lection cannot be cut Copy 3 C CTRL C Copies selected highlighted text or data and stores it on the clipboard so it can be pasted elsewhere This menu item is dimmed if no selection has been made or if the selection cannot be copied Parts of an experiment can be copied within the same experiment or to another application using the Copy and Paste commands You may also use this command to copy information from SoftMax Pro into other programs Paste 3 V CTRL V Pastes information from the clipboard just to the right of the insertion point in the document Can also be used to import tab delimited data into SoftMax Pro This menu item is dimmed if noth ing has been copied to the clipboard Clear Clears or erases highlighted text or data without storing it on the clip board This menu item is dimmed if nothing has been selected You can remove one or more columns from a Group table or text or formu las from a Notes section by highlighting the column or text formula and then choosing Clear from the Edit menu Columns and text or for mulas can also be removed by highlighting them and pres
321. ed Interprocess Messaging Features New and Modified Features for Robotic Integration 1 Expanded COM ports selection 0 9 2 Extended communication response timeout value and removed the time out warning dialog during remote command operation 3 Added SetFolderAs XXX command User can set the current protocol directory using this remote command User has to specify the directory or file path Example SetFolderAs c SoftMax Pro 3 1 2 Basic Protocols 4 Added a parameter to the ExportAs XXX command append data to an existing export file and import the file as a single document T The ExportAs command without the a attribute overwrites any exist ing file without warning However user can choose to append export data to the end of an existing export file by specifying the a attribute In addition this export file can then be imported and all data can be viewed as a single document file Please note that a single space is required between the attribute and the following directory and file string XXX Example ExportAs a c SoftMax Pro 3 1 2 exportfile txt SoftMax Pro 3 2 Update Specification FlexStation Features SoftMax Pro User s Manual e Supports all the features of the new FlexStation instrument including a new read mode also called FlexStation New tabs are included in the Instru ment Settings dialog box for the new read mode e The Plate section for FlexStation instruments shows which w
322. ed as a mathematical operation Replicate Samples and Samples in a Series You can assign the same group or sample definition same sample name and description within a group to multiple wells to create replicates For example you might want to read standards in replicate to ensure that anomalies can be excluded prior to generating a standard curve Replicates can be created using the Assign button or with the series function Creating a series is faster than assigning replicates individually The series option can be used as long as the replicate values conform to a pattern that can be described by a mathematical operator multiply by 2 or add 5 for example A series is defined in one direction starting from left right top or bottom and therefore samples must be arranged sequentially in either ascending or descending order To create a series do the following 1 In the Template Editor highlight the wells to be part of the series The wells must be in the shape of a rectangle Experimentitl Plates rop Dome EET smesso o Concentration t Series Group Name Button Pull Down Menu Sample Name Box and Selected Wells Units Figure 4 27 Template with Wells Selected for a Series 2 Click the Series button to open the Series dialog box 4 30 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Series Start From First Sample S
323. ed object Clipboard A temporary storage location within the operating system used to transfer data within and between files and applications Compound and Tip Columns FlexStation only A selected tip column and compound column are assigned for fluid transfer to a spceific assay plate column Compound Source FlexStation only Compounds are transferred from the compound source plate to the assay plate Compound Transfer FlexStation only Microplate fluid transfer from the compound source to assay plate Concentration The amount of a substance present per unit volume Used as the default sam ple descriptor for a series of samples SoftMax Pro User s Manual B 3 Appendix B Glossary of Terms Control A sample that is run as an internal check of the assay The control has a pre dictable or known assay result against which other results can be compared TH Control Menu Box Windows The icon that opens the Control menu for the window It is located at the left of the SoftMax Pro title bar The Control menu can be activated by clicking in this box with the mouse or by pressing ALT SPACEBAR Copy A function common to most software applications that duplicates the selected text or item and places this duplicate on the clipboard The copy function is enabled by pressing 3 C on the Macintosh and CTRL C in Windows Copy and Paste A method of copying selected text or graphics from one location and pasting them to a di
324. ed within the file are listed on the left side of the Source field in the Plot X dialog box The first group in the file is automatically selected Other groups can be selected by highlighting them When a group is selected the pull down menus in the X axis and Y axis subfields change to show the group columns that are available for graphing Group columns can be assigned for both the values to be plotted and any error bars to be included for both axes button for either the X axis or Y axis opens a dialog box which allows you to enter formulas To plot a summary enter the summary name in this dialog box Formulas can be plotted for comparison with other data For example if the data is assumed to follow a specific model the model formula can be entered as a plot and then the data can be chosen for another plot within the same graph You can also use this feature to plot Kinetic or Spectrum data in 8 45 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 1 k 8 46 Graph sections For example to plot Kinetic data from well A1 in a Graph sec tion you can enter the formula A1Lm1 for the Y Axis and the TimeRun accessor for the X axis The entered formulas are also shown in the Source field of this dialog box The size color and style of the icon used for a plot can be selected on the right side of this dialog box Four sizes nine colors and nine styles are available from drop down
325. eee es 10 53 Plate Section with Data from Saved File 0 0 cee eee eee 10 54 Display Diglot DOK 34222242 etonws vanees neues EEA UEM NO Riu dox P dep hs 10 55 Display Options for Reduced Displays 0 0 0 ccc eee esses 10 55 Display Dialog Box Updated with Low and High Limit Settings 10 56 Plate Display with Data Displayed as Gray Scale 00 eee eee 10 56 The Group Section for Data sssr ts sonet oropdex tra oy sd aded n qi tees 10 57 A Portion of the Data Group Section with Formulas Shown by Pressing the CTRL Shift Keys 0 0 cece eee e eee eee 10 58 Data Group Section with Formula Shown for Highlighted Column CO Activa hon serrer Hones ooo en Ord eb URS IUE ESO uem EE dpi 10 58 Sizing the Data Group s Concentration Column Wider uuu 10 59 Columns Before and After Autosize esee 10 60 CalculabonDidos DOX2eaaa vues d uat pahsdci dod d dique de Hu S qa ud oup anes mee 10 60 New MaxValue Column Appended to Group Section 0 10 61 Summaries in the Standards Section 0 eee eee cee eens 10 62 Calculation Dialog Box from Summary in Standards Section 10 62 Standard Section Before and After Hide Replicates 00 10 63 Graph Section 4 Paramter Fit Applied 0 0 eee cee eee 10 64 Graph Options Dialog Box 1 1 0 0 cece nnn eens 10 64 Cluster Bar Graph Displayed 0 2 cece eee eese
326. eena es 8 45 Formulas in 5oftMax PrO a2 cciceecerense seh serene tee om hn tds 8 64 viii SoftMax Pro User s Manual SoftMax Pro User s Manual Chapter 9 File Management and Printing 0 0 e eee eee 9 1 File Creation and Management 0 0 0c eee ee ee eee eee 9 3 Dera Proto Ol aserra teases irren 9 3 Locating Protocol and Data Files 0 008 9 5 Creatine New P368 ron koh 65d 1E Pede qud Pad eR 9 5 OPa E O Er Pm 9 6 Savine Files Manually uad 0e irinak a rriei qt eco ad 9 8 acci EE E EEEE 9 9 PAOD eee ee ee ee EE pd dde ee 9 11 The Printed Report 0 0 cece cece teens 9 12 Customizing the Report 00 cece eee eee eee ee 9 12 The Print Dialog Box eeeeeeeeee eee 9 14 Exporting Importing Information 0 e eee ee eee 9 16 EX DOUG Dali rade aie ad ahi Hea ne eee ke owe ee 9 16 Importing Data from Analyst Acquest ScreenStation cuo PU Saree ee ee 9 21 Importing and Exporting Templates 0 9 24 Exporting Graphs 0 0 0 eee eee eee 9 25 Copying and aS qe ou ir kemia mat heads it 9 25 Pile TP MOlCCHON PP e 9 29 Password Protection 0 0 cece ee een 9 29 Chapter 10 Tutorials 220m 10006400 Dar ERROR dca dE dee 10 1 IM OCUCHOR Rc UR 10 3 Tutorial 1 Quantitative Endpoint Assay Tutorial 10 4 Hard and Soft Parameters 00 c eee ee eee 10 4 Description of the
327. elength Priority During a Reading SoftMax Pro User s Manual Depending upon the type of instrument connected to the computer or chosen in the Reader Preferences a priority is given to reading by column or wavelength It is most important to understand this priority with multi wavelength readings In column priority an instrument will read the entire microplate or chosen number of strips at the first wavelength and will then go back and read the microplate at the second and subsequent wavelengths In wavelength priority the instrument will read all wavelengths for the first column of wells in the microplate and will then read all wavelengths for the second column etc 0 35 Chapter 5 Reading Using Fluorescence or Luminescence Instruments The table below shows the priority settings by instrument for the different read modes Table 5 3 Column vs Wavelength Priority SpectraMax Gemini Column Wavelength Column Fluorescence Readers Data Display During a Reading The values read by the instrument will appear in the data display of the Plate section as they are received from the instrument in real time Depending upon the read mode Endpoint Kinetic Spectrum or Well Scan the number of wavelengths the plate type and the display options selected the data dis play will show one or more numbers or plots You can enlarge the display of one or more wells while the a Kinetic or Spec trum reading is in progress
328. ell Standard clrbtm Wells To Read Read area amp 1 H10 Compound Source Beckman 96 2 3mL Figure 10 47 Instrument Settings Dialog Box Showing Automix 10 42 SoftMax Pro User s Manual Chapter 10 Tutorials AutoCalibrate Check the setting for AutoCalibrate the default Once is fine and does not need to be changed Assay Plate Type The default setting for Assay Plate Type 96 Well Standard clrbtm is fine and does not need to be changed Wells To Read In this Tutorial we will not be reading all the wells in the plate Click on the Wells To Read heading on the left pane to show the grid of wells on the right Click in well A1 and drag the mouse cursor diagonally to well H10 Col umns 1 through 10 should now be highlighted shown in blue indicating that these wells will be read by the FlexStation When you are finished high lighting wells the dialog box should appear as shown below m Instrument Settings E X Endpoint Kinetic Spectrum Well Scan Options Wavelengths Ex Em Auto Cutoff On 485 525 515 Sensitivity Readings 3 PMT High Timing Wells To Read Time 200 secs Interval 2 secs Reads 101 Minimum Interval 1 28 secs Automix Before Off Between Off AutoCalibrate Once Assay Plate Type 9B Vell Standard clrbtm Wells To Read Read area amp 1 H10 Compound Source Beckman 96 2 3mL Cancel Figure 10 48 Instrument Settings Dialog Box Showin
329. ells have been selected for transfers small color coded triangles in the corners of the wells show first and subsequent transfers e Information about the transfer settings and compound sources is shown beneath the Plate display e Because intervals between readings are not predictable in FLEX mode Soft Max Pro collects and stores a time tag for each reading made in FLEX mode This time tag is used in analysis and graphing of the resulting data e When SoftMax Pro is connected to a FlexStation instrument additional items are available in the Control menu Open Close Tip Drawer Open Close Compound Drawer Set Air Gap Allows you to set the distance from 0 to 200 milliliters between the tip of the pipette and the bottom of the plate well e Bottom Read allows the FlexStation instrument to take readings upward from the bottom of a microplate instead of downward from the top A 7 Appendix A SoftMax Pro Version Specifications 1 3 New Accessors e New accessors have been created to accommodate the new instrument set tings included for FLEX mode These include accessors for transfer vol umes transfer rates transfer timing pipetter heights and source types for each transfer Accessors for transfer areas source areas and tip types are also available Checksum e The checksum feature has been activated for the FlexStation instrument to check for accuracy of received data Import Export e SoftMax Pro can now exp
330. en lost during this read Also one or more points were reported as saturated m 5UFTmax Pro x One ar mare points may have been lost AN during this read Also one or more points were reported as saturated Please check your data NON This error appears only during Kinetic or Spectrum mode with fluo rescence instruments This error message appears if a specific timepoint in a given well for a Kinetic run or a specific wavelength in a given well for a Spectrum scan is saturated and points are also missed see One or More Lost Points above Check the individual wells for saturation If the saturation occurs at the time or wavelength of interest you will need to reread at a lower PMT setting If the saturation does not occur in the time segment or wavelength of interest you may ignore the message and proceed with data analysis For points missed see the earlier section One or nore points may have been lost during this read on page 11 6 for information on how to find lost points One or more points were reported as saturated SoftMax Pro User s Manual m SOFT max Pro x One ar mare points were reported az saturated Please check your data NOINA This error appears only with fluorescence instruments during Kinetic or Spectrum mode A specific timepoint in a given well for a Kinetic run or a specific wave length in a given well for a Spectrum scan is reported as saturated Check th
331. ens the Reduction dialog box By default the ranges on the enlarged plot are set to the limits defined in the Reduction dialog box and the information included near the bottom of the window is based on the reduction settings currently in use You may change these settings wavelength combination and type of reduction used minimum and maximum OD limits lag time and end time and number of Vmax Points for a cuvette whether or not to use a template blank absolute values or to show the data as absorbance or percent transmittance More information about the reduction options can be found later in this chapter Changes to the reduction settings made within well graphs will be applied to all wells or cuvettes in the Plate or CuvetteSet section When viewing Raw data you can scale the graph to see data outside the reduction limits line s are included on the graph indicating the limits and the area outside of them will be shaded slightly This view should assist you in determining the best limits for the reduced data e Show Raw Show Reduced button This button toggles the Well Graph between views of raw and reduced data When the Well Graph is opened the initial view depends on the Dis play settings for the Plate section If raw data is being displayed in the Plate the initial view in the Well Graph will be raw data and the button will be set to Show Reduced If reduced data is being displayed in the Plate section the initial view in the
332. ent an OKRead text string will be returned after the read is com pleted B The user does not wish to receive a completion message Use SendMessage combined with WM_SETTEXT for messages that do not require a completion message The advantage of this approach is that it may be universally applied to Visual C and Visual Basic modules Excel macros etc 3 Remote commands that DO return data There are three remote commands that return data ReturnStatus ReturnTim ing and ReturnData See SoftMax Pro Commands in this chapter for details It the caller has enabled queue operation the command ReturnStatue is han dled immediately and not queued as are other remote commands Data is returned in one of two ways A After sending the remote command using the WM COFYDATA message the caller can look for return data through an asynchronous Windows message inside a Win32 COPYDATASTRUCT type data packet An asynchronous window message is returned as a window callback typically an OnCo pyData handler B After sending the remote command using the WM SETTEXT message the return data will be available immediately on the clipboard The advantage of this approach is that it may be uni versally applied to Visual C and Visual Basic modules Excel macros etc SoftMax Pro User s Manual Programmer Notes e COM ports 1 9 can be used for robotics purposes Molecular Devices rec ommends using COMI or COM2 These are typical
333. ently exists the new Plate section will use SoftMax Pro de fault settings New CuvetteSet Creates a new CuvetteSet section below the active section The new CuvetteSet section will be a copy of the active CuvetteSet sec tion or if no CuvetteSet section currently exists the new CuvetteSet section will use SoftMax Pro default settings New Graph Creates a new Graph section below the active section and brings up the Plots dialog box allowing you to select the graph options 3 30 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Control Menu Control Instrument Setup Read R Ref Close Drawer Incubator Calibration Open Tip Drawer Open Compound Drawer Set Air Gap Recover SoftMax Pro User s Manual Instrument Setup This command is available only if a Plate section or CuvetteSet section is active Choosing Instrument Setup opens the InstrumentSettings dialog box which allows you to enter the mode for the run Endpoint Kinetic Spectrum Well Scan or FLEX the wave length settings Automix parameters pre read blanking strips PathCheck AutoRead timing for Kinetic runs Speed Read for Spectrum scans read type and plate type NOH Notalloptions are available for all instruments The options that are visible depend on the type of instrument that is connected to the com puter or selected in the Preferences Button Equivalent Setup in the Plate s
334. er of fields of data that you have copied does not match those required by the setup of that Plate section or CuvetteSet section For Plate section data the format of the copied information must match the Instrument Settings of the target Plate section and the settings chosen in the Export Format in the Preferences Check that all the required fields are present and that you have highlighted all of these fields prior to copying the data and try pasting again SoftMax Pro User s Manual Chapter 11 Troubleshooting Instrument communication problem Response too late gw SOF Tmax Pro Instrument communication problem Response too late This error appears when the cable connection between the computer and the instrument is not functioning properly Clicking will stop the run This error appears if the cable has been disconnected but it could also occur with a bad cable serial port or communications board Recover will reset FlexStation m gt OF Tmax Pro x Recover will reset FLE station ou must manually remove the pipette head and dispense any liquids before resetting Failure to do sa may lead to serus damage Please refer ta the FLE station manual before proceeding 5s Continue Wess This report appears with the FlexStation instrument only This message appears after you choose Recover from the Control menu Choosing the Recover command may be necessary after a power failure or after shu
335. er s Manual SoftMax Pro allows you to print several types of information In addition to printing a copy of the template you can print the active section the sections that have been designated to be included in the report or all sections The default setting in SoftMax Pro is for all sections to be included in the report You can exclude sections from printing as described in Chapter 9 File Management and Printing Otherwise choosing Print from the File menu and choosing Print report will print all of the sections that are designated to be included in the report regardless of whether or not they are open A standard Print dialog box will appear the appearance of this box depends upon the type of computer and printer you are using Select the appropriate settings for your printer and then click Print or OK Printer General 2531 Print Copies 1 Pages amp All Qrrm To Cancel Paper Source O EE Destination e p visi All QFirstfrom Auto Select w Printer i wr Ofte mals ani S kf Date and Time xf File Name kf Page Numbers Q Print All Print Report Figure 10 82 Print Dialog Box for Macintosh Print eee 2 x Printer Name EPSON Stylus COLOR 980 Properties Status Ready Type EPSON Stylus COLOR 980 Where USBOO1 Comment Print to file Print range m Copies Al Number of copies fi
336. eres 10 65 Detail from Graph Section Showing 50 Location on Plot 10 66 Calculation Dialog Box for EC 50 Summary Formula 10 66 Print Dialog Box for Macintosh 0 cece eect eens 10 67 Print Dialog Box for Windows Epson Stylus COLOR 980 shown 10 67 Example or an Alert VIeSSdBO sccadvrteit cpenbes REA UE ua PX ARE rpg 11 3 About This Computer Dialog Box seeeeeeeeeee eee 11 16 Soft Max Pro InfO Dialog BOX oen eren rmm trm nm e eed 11 17 SoftMax Pro Remote Command Interface lees 12 3 VVeW Scam Edr ODNON PR A 4 SoftMax Pro User s Manual Tables Table 2 1 Table 3 1 Table 3 2 Table 3 3 Table 3 4 Table 3 5 Table 4 1 Table 4 2 Table 4 3 Table 5 1 Table 5 2 Table 5 3 Table 7 1 Table 7 2 Table 7 3 Table 7 4 Table 8 1 Table 8 2 Table 8 3 Table 8 4 Table 8 5 Table 9 1 Table 9 2 Table 9 3 Table 9 4 Table 9 5 Table 10 1 Table 10 2 Files that Are Installed 2 oo2sd22ngtapos peo ademas tusdatavedx vede aq dide 2 10 Notes Section Menu Commands and Tool Bar Buttons 00 3 14 Plate Section Menu Commands and Tool Bar Buttons 004 3 16 CuvetteSet Section Menu Commands and Tool Bar Buttons 3 18 Group Section Menu Commands and Tool Bar Buttons 04 3 20 Graph Section Menu Commands and Tool Bar Buttons 0000 3 22 Features Available in
337. erial port set to modem in the SoftMax Pro Preferences dialog and the switch box set to communicate with the MDC Microplate System instrument to access the Chooser or Preferences dialogs refer to To Switch from Printing to Reading below Configuration for Printing The computer SoftMax Pro for Macintosh and switch box must be config ured as follows in order to print from SoftMax Pro AppleTalk active in the Chooser dialog serial port set to modem in the SoftMax Pro Preferences dia log and the switch box set to communicate with the printer to access the Chooser or Preferences dialogs refer to To Switch from Reading to Printing below Connecting to the Switch Box These instructions are for connecting to the A B switch box available from MacWarehouse 800 255 6227 part number BND0199 DIN 8 A B switch box Attach one end of the 8 pin DIN to 8 pin DIN serial cable provided with the switch box to the single 8 pin DIN serial port of the computer Attach the other end of the cable to the input port I O of the A B switch box Attach one end of the 8 pin DIN to 8 pin DIN serial cable provided in the SoftMax Pro software package to one of the two output ports A or B of the A B switch box Attach the other end of the cable to the MDC Microplate System instrument Attach one end of the 8 pin DIN to 8 pin DIN AppleTalk cable to the remaining output port of the switch box Attach the other end of the cable to the AppleTalk
338. error bars relative to a particular axis are the same as those available for displaying data on that axis any column from that group that contains numerical information not columns with text To display error bars click and hold the box next to Error Bars in the Plot X dialog and drag to select one of the choices in the menu To disable error bars choose No Error for the X and or Y axis Error bars show both positive and negative error Depending on the range set tings for the graph portions of the error bars may not be visible Curve Fitting No Fit selected SoftMax Pro User s Manual When you first create a graph of the data it does not have a fit associated with it The graph might appear something like this v 7 Graph Fit Graph 60 concentration Plot 1 Group 1 concentration vs Mean value Figure 7 42 Graph with No Fit 7 53 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 1 B 54 You can fit any plot to one of nine curve fitting algorithms Linear Semi Log Log Log Quadratic 4 Parameter logistic Log Logit Point to Point Expo nential and Cubic Spline These selections are shown in the Fits pop up menu in the Graph section toolbar click on the box next to Fit Typically a standard curve refers to the curve fitted to the plot of concentra tion versus mean value for the Standard group Once you have selected a Fit type SoftMax Pr
339. es menu dialog box Description Tool Bar Button Opens the Calculation Create Summary in the dialog box allowing you 24 Notes menu 5 5 to create a summary Edit Summary in the Notes menu or double Opens the Calculation click the summary dialog box allowing you pointer changes to dou to edit the summary ble headed arrow Shows hides the calcu lations used to derive summaries l Includes excludes sec Printer icon j tion from printed report Show Hide Formulas in the Notes menu 3 14 SoftMax Pro User s Manual Jil Chapter 3 SoftMax Pro Overview Plate Section Tools v Ed Peteri Z aj Plate 1 Data Display Spectrum Fluorescence Excitation Cutoff EmStart Stop Automix Off Calibrate On PMT Auto Instrument Settings H C mtl oe eR nd ot o dz well 6 Reduction Settings Wavelength Combination Lrm1 dii dosis Plate Blank Used Start 350nm End 75 nm RFU Fin 0 RFU Mas 20000 Flate Last Fead B 42 AR 6730799 Figure 3 11 Plate Section The Plate section is used to collect data from the instrument define how the data is displayed and what the initial reduction of the data will be In addi tion the Template Editor in the Plate section is used to create a map of the contents of the microplate Choosing a particular function or command in the Plate section can be done in several ways You can use the mouse to click on a butt
340. es section text can be copied from programs other than SoftMax Pro and pasted into a Plate section Cuvette Set section or Notes section For Plate section data the format of the copied information must match the Instrument Settings of the target Plate section and the settings chosen in the Export Format in the Preferences Pre read data cannot be pasted into a Plate section If there is a mismatch between the data to be pasted and the target section SoftMax Pro will attempt to paste the data but if it cannot do so an error message will result an example of such an error message for a Plate section is shown in Figure 9 16 After data is pasted into a Plate sec tion the information to the right of the display will show imported data 5 Pasted Text doesn t match plate settings Figure 9 16 Error Message Generated When Pasting Mismatched Data If you have created a template for a Plate section prior to pasting data numbers will be shown only in the group s you have created in the template Data in other wells will not be reported but will be available if you alter the template Clearing the template or assign ing groups to all wells in the plate will show the complete data set Templates for CuvetteSet sections do not change the way the data is shown in the display all cuvettes will show data regardless of whether or not the template has only certain cuvettes assigned to groups SoftMax Pro User s Manual Chapter 9 File Mana
341. es this reading as a reference for all cuvettes in the CuvetteSet section Template Blanks are created by entering a Blank group in the CuvetteSet sec tion template After a reading the average of the blank values obtained from the Blank group is subtracted from all cuvettes Group Associated Blanks are created by placing blank cuvettes within a group in the template The values of group blanks are averaged and sub tracted from the values of the cuvettes within that group only Blanking Plate Section Three types of blanking are available in the Plate section Pre Read Blanking Plate Blanking and Group Blanking Pre Read Blanking subtracts background signal caused by the microplate itself as well as by solvents reagents or samples The individual blank read ing from each well can be subtracted from the final reading from the same individual wells choose Pre read plate from within the Instrument Settings dialog box This is usually used for measurement in the deep UV or for path length correction NOJA Pre read blanking is not available with all instruments and also is generally not used with Kinetic assays If used with Kinetics it will complete a pre read Kinetic run and then subtract these values on a SoftMax Pro User s Manual Appendix B Glossary of Terms point by point basis from the normal Kinetic run Plate Blanks are created by entering a Blank group in the template After a reading the a
342. escence RFUs C Luminescence RLUs Figure 5 8 Instrument Settings for a Well Scan Reading You can choose up to four wavelengths Read Type Choices for the read type are fluorescence luminescence or time resolved flu orescence Also included in this group of settings is bottom read To select a read type click the appropriate section in the Instrument Settings dialog box All read modes are available for all read types Two types of scans are possible when either fluorescence or time resolved flu orescence read type is chosen you can select a fixed wavelength for excitation and scan emission or vice versa More about this can be found in the follow ing section Wavelengths Fluorescence The light emitted by certain substances resulting from the absorption of inci dent radiation To measure fluorescence accurately it is necessary to reduce light scatter The governing equation for fluorescence is Fluorescence extinction coefficient concentration quantum yield excita tion intensity pathlength emission collection efficiency Luminescence The emission of light by processes that derive energy from essentially non thermal changes the motion of subatomic particles or the excitation of an atomic system by radiation Time Resolved Fluorescence Most fluorescence substances are not suitable for this type of reading How ever the fluorescence emitted by lanthanide dyes is delayed long enough to SoftMax
343. escriptor Dilution Factor r Units for Sample Descriptor Column Format Setting Choices are Column Format nnn y Basic Standards Unknowns Unknowns Dilution Figure 4 21 Group Settings Dialog Box for the Group Named Patient 1 The name sample descriptors and column format can be changed for the group that you are editing When you edit a group you will notice that the information shown for the Column Format in the Group Settings dialog box changes to show the name of the original group used to define the group columns see Figure 4 22 Group Settings Name Standards FD Sample Descriptor Concentration Units Imm F When you are editing Group Settings rather than creating a new group the word original precedes the Column Format Figure 4 22 Groups Settings Dialog Box Showing Original Column Format 4 27 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 1 k 4 28 NISWEGNR Changing the column format setting will cause the columns in the group to revert to their original defaults as shown in Table 4 2 on page 4 26 If you have added columns or changed column information within the existing section for the group you are editing these changes will be removed The Button Allows you to deselect or clear wells in the map of the microplate Highlight the wells you wish to clear and then click the Clear button The button functions the same way
344. et w Plot symbols Im Pots ew pee m X Concentration Standards Y Mean Valued standards m Errors Mo Error Y Errors Mo Error Figure 10 40 Graph Options Dialog Box This dialog box allows you to choose rename the graph choose the type of graph that will be shown and to format the text used in the graph You can add or remove plots click the or Delete buttons and you can also edit existing plots Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using provide more information regarding the graphing options that are available 10 33 Chapter 10 Tutorials Step 10 Print a Report SoftMax Pro allows you to print several types of information In addition to printing a copy of the template you can print the active section the sections that have been designated to be included in the report or all sections The default setting in SoftMax Pro is for all sections to be included in the report You can exclude sections from printing as described in Chapter 9 File Management and Printing Otherwise choosing Print from the File menu and choosing Print report will print all of the sections that are designated to be included in the report regardless of whether or not they are open A standard Print dialog box will appear the appearance of this box depends upon the type of computer and printer you are using Select the appropriate settings for your printer and then cl
345. ette Display Display Template B Template Graph Reduction Reduction Mask Copy Template Paste Template Graph Mask Copy Template Paste Template i Display Graph Mask Copy Template Paste Template Export Template Import Template Section Mame New Window Display Graph Mask Export Template Import Template Section Name Mew Window Export Template Import Template Section Name New Window Copy Template Paste Template Export Template Import Template Section Name New Window Figure 4 33 Export and Import Template Commands in the Plate and CuvetteSet Menus Template files can be prepared outside of SoftMax Pro and then imported as needed when ready to run samples How your particular templates should appear depends on the type of samples you will run whether or not you will run them in replicate etc An example template containing two wells with blanks one column of standards and the rest of the wells containing unknowns with replicates and serial dilutions is shown below as it would appear in SoftMax Pro followed by a portion of the exported version of the same template as seen in a spreadsheet program SoftMax Pro User s Manual 4 37 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 4 38 1 2 BL StaQt dee Lee ee ee m Figure 4 34 Template Example Showing Two
346. ew and edit the formulas associated with the various columns or create new columns containing new formulas The data shown in columns from one group can relate to or contain infor mation derived from columns in another group You can also create sum maries which consist of formulas and text e Notes sections allow you to enter summaries which consist of formulas and related text Use of formulas in SoftMax Pro is described in a separate manual The SoftMax Pro Formula Guide 67 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments t 7 68 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments IDECOCIU CHO eiae etes dnte ES emit Pop redi ies sesto qe iud Ds 8 3 Initial Data Display and Reduction 0 00 00 00 000088 8 4 hie Data Display swt diene n PIER TPSIDPRMRPHPUE eines sag ESSI 8 5 The Display Dialog Bots snp cukier dey queer EP eee SO 8 6 Graphine Well c22aceorauuseen tive okt peas Gees perso ee 8 14 Masking Welle geaterna id nd td RU de cy X PES be 8 18 Data Reductob secs Rena tid ome ty ee T ur Vei yw ecran ie ns 8 19 BRO DOU bosco wu Viren C tA EPI P Et es viste 8 21 KINCUC eceos dem eds etis exte UU E VE Hd a 8 21 DPC CHUN M D PR sen aed hea pane er Sees Ges 8 29 Well ocan oe opea dor ass cy eU eo PLI USE ves PME 8 30 PUEA TE 8 31 Custom Reduction Formulas esses essen 8 35 Recalculation OpDODS 921 mq mod e Pe
347. ew section can be modified as desired Ness dIncubator control is an instrument setup parameter that is not found in the Instrument Settings dialog box 5 36 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments FLEX Mode Run Time secs overs Automix mtn tnennee enitn 1 Interval secs Before First Read secs Number of Reads 34 B Minimum Interval 1 52 secs Minimum Run Time 34 secs AutoCalibrate Fast Normal Precise JV AutoCalibrate 8 Assay Plate Type Wavelengths EL Select an assay plate type 96 well plates 96 Well Standard Fi 96 Well Greiner blk clrbtm 96 Well Greiner clear 96 Well Costar blk clrbtm 96 Well Costar clear 96 Well Falcon clear 384 well plates 384 Well Standard 384 Well Greiner blk clr 384 Well Greiner clear n Excitatpn Emission E N M wa Instrument Settings Ep x Endpoint Kinetic Spectrum FLEX Options Wavelengths Ex Em Autd utoff On 485 538 Jo Sensitivity Readings 6 PMT Hig Timing Time 300 secs Interval 9 secs TS E S weenn r Number of Wavelepaffis Excitgfon Emission IV Auto Cuttoff s v bse v 57 Lint Reads 34 f i Automix Select a compound plate type Before Off 12 Column Plates Between Off Beckman 140504 AutoCalibrate Soar ar Costar 3
348. f Last Read Ex 355 Calibrate On 7 14 99 Em 612 PMT Auto 7 59 4M Cutoff 610 Reads Well 6 Auto Cutoff Wavelength Combination Lrn1 v C3 Plates a aw data in 1000 s Plate 1 1 3 5 7 9 11 13 15 17 19 21 23 2 4 6 8 10 12 14 16 18 20 22 24 41 1 61 1 81 2 21 2 61 3 11 3 71 4 41 5 31 6 41 7 71 81 2 21 2 61 3 11 3 71 4 41 5 31 6 41 7 71 9 41 11 4 151 3 11 3 71 4 41 5 31 6 41 7 71 9 41 11 4 13 8 16 8 171 4 41 5 31 6 41 7 71 9 41 11 4 13 8 16 8 20 424 131 6 41 7 71 9 41 11 4 13 8 16 8 20 4 24 9 30 3 36 9 171 9 41 11 4 13 8 16 8 20 4 24 9 30 3 36 9 45 1 54 9 1 4 13 8 16 8 20 4 24 9 30 3 36 9 45 1 54 9 67 0 81 8 5 8 20 4 24 9 30 3 36 9 45 1 54 9 67 0 81 8 90 8 122 51 1 81 2 21 2 61 3 11 3 71 4 41 5 31 6 41 7 71 9 41 121 2 61 3 11 3 71 4 41 5 31 6 41 7 71 9 41 11 4 13 8 111 3 71 4 41 5 31 6 41 7 71 9 41 11 4 13 8 16 8 20 4 I 41 5 31 6 41 7 71 9 41 11 4 13 8 16 8 20 4 24 3 30 3 41 7 71 9 41 11 4 13 8 16 8 20 424 30 3 36 9 45 1 1 41 11 4 13 8 16 8 20 4 24 9 30 3 36 9 45 1 54 9 67 0 3 8 16 8 20 4 24 9 30 3 36 9 45 1 54 9 67 0 81 8 99 8 0 4 24 9 30 3 36 9 45 1 54 9 67 0 81 8 99 8 122 149 indpoint Lm Automix Off Last Read 405 Calibrate On 7 16 99 450 PMT Auto 7 44 AM Fluorescence 7 005 7 705 10 305 11 405 8 505 9 405 110 305 11 405 12 505 13 805 10 30511 1 405 12 505 13 805 15 205 16 805 5 805 6 405 7 005 7 705 8 505 9 405 10 205 11 405 12 505 13 805
349. f a value other than 0 is shown highlight the existing value and enter O in this box Click the Assign key or press either the Tab or Return key on the keyboard to assign this concentration to the selected wells in the template You can quickly check the concentrations you have assigned to the template by pressing and holding the CTRL Shift keys Macintosh or Win dows This causes the display to show only group names and values assigned to specific wells m Experiment 1 Plate 1 E X mum Sri J ase Clear amp oncentration n uci 1 2 3 4 5 T B J 10 12 Blank BL BL Standards TES eH ST i Print Cancel LK Figure 10 6 Template showing First Standards Concentrations C Now select wells C1 and C2 Standards is still showing as the default group name but a new sample descriptor is created automatically St02 so all you need to do is assign a concentration value of 0 1 ug ml to these wells After entering the value click the Assign box or press the Tab or Return key to assign this value to the selected wells D Atotal of seven standards will be assigned in duplicate in this template You can use the Series function to assign the next four standards Select wells D1 D2 through G1 C2 Click the Series button In the dialog box that appears leave the default values for the sample descriptor Std03 starting from the top and the concentration in ug ml Enter 0 25 for the
350. f connect Points wf Plot Symbols Figure 8 9 Graph Options Dialog Y Axis Settings button not found in Well Scan mode Opens the Well Graph Axis Settings dialog box for the particular axis allowing you to set Auto Range parameters choose the minimum and max imum values for the selected axis cause the axis to be auto ranged add or remove gridlines and add or remove axis tick marks When a Well Graph is opened the default settings are to show the reduction limits for that axis with Auto Ranging off and gridlines and tick marks selected Only the Well Graph being viewed is affected by changes in these dialog boxes Done Closes the Well Graph dialog box I piate 1 well Graph Reduced ioj x 6500 X I
351. f the Data Group section Shown there in large format type is a summary that states Summary 1 EC 50 0 03101 Click this summary to highlight it If you look up in the gray tool bar at the top of the window you will see the formula used to create this summary is shown there Summary 1 InterpX Plot 1 Graph 1 50 Let s examine this for mula further to understand how it was derived Formulas can be simple or quite complex and are composed of elements that perform mathematical operations statistical analysis functions and or refer to other elements contained within SoftMax Pro Complete information about the possible choices for creating formulas can be found in the Formula Refer ence Guide The formula from the Summary in the Data section of this Tutorial file uses a function InterpX which interpolates data from the curve fit in the Graph section The InterpX function returns the X values of the speci fied plot interpolated using the Y values and curve fit The complete formula is written in this manner InterpX Plot Graph Yvalues In this particular summary formula Plot 1 Graph 1 describes the plot from which the data is derived Yvalues in this example is 50 which 10 65 Chapter 10 Tutorials describes the number to be interpolated So looking at the graph the Y value at 5076 activation appears at an approximate concentration of 0 031 1 Concentration Figure 10 80 Detail from Graph S
352. fferent location in the same file or into another file which could be in an application other than SoftMax Pro Analyst System Molecular Devices Analyst System line of instrumentation formerly from LJL BioSystems is specifically designed to address the microplate based assay detection needs of Life Science and Drug Discovery laboratories Cubic Spline Curve Fit This curve fitting option generates a fit to a cubic equation between each pair of data points The cubic equations are computed such that these equations and their first two derivatives are continuous everywhere More information regarding curve fits can be found in Chapters 7 and 8 Curoe Fit Specifies the type of curve linear logarithmic quadratic and so on that is applied to the data points in a plot The curve is a graphic representation of the mathematical relationship between the data points in the plot More infor mation regarding curve fits can be found in Chapters 7 and 8 Cutoff A filter used to condition the light entering or exciting the monochromators In automatic mode the instrument sets the cutoffs automatically based upon the wavelength s chosen for reading with some read modes and types how ever you can choose a different filter wavelength manual setting for the emission monochromator CuvetteSet Section SpectraMax Plus and Plus only The CuvetteSet sections determine how the cuvettes will be read display data as it is acqu
353. formance of SoftMax Pro version 3 0 for Macintosh cannot be guaranteed when using this model When running SoftMax Pro the single serial port Macintosh can either com municate with the MDC Microplate System instrument or print through the single serial port but both printer and instrument cannot remain connected to the serial port at the same time To avoid uncabling the instrument in order to attach the printer you must either print across a network using the ethernet port of the computer or the instrument and printer can be connected to an A B switch box one 8 pin DIN input port with two 8 pin DIN output ports available from MacWarehouse 1 800 255 6227 part number BND0199 Din 8 A B switch box This switch box can then be attached to the single serial l FlexStation and SoftMax Pro both boot up using a baud rate of 9600 to to com municate over the serial line SoftMax Pro boosts the baud rate to 57600 when FlexStation is reading in FLEX mode When the read is finished SoftMax Pro automatically reduces the baud rate to 9600 again In order to communicate at dif ferent baud rates SoftMax Pro sends a command to the FlexStation to change its baud rate also Communication is lost if the baud rates don t match Under nor mal operations it shouldn t be necessary to change the communication speed Should communication be lost and other measures do not restore it you may attempt to restablish communication by resetting the baud rate manually u
354. forms an air reading at all available wavelengths and stores those values in the non volatile instrument memory NVRAM For plate readings these air calibration values are used whenever a Spectrum reading is performed using the Speed Read option thus reducing the time it takes to complete a Spectrum reading For cuvette readings the air calibration values are used whenever a reference reading has not been taken or when the cuvette is read using the control panel of either the SpectraMax Plus or Spec traMax Plus Molecular Devices recommends that you update the air reference information that is stored in NVRAM periodically This is particularly important if you are using Speed Read reading cuvettes without a reference or running Kinetics in microplates with read intervals that are too short to allow autocalibration Column vs Wavelength Priority During a Reading Depending upon the type of instrument connected to the computer or chosen in the Reader Preferences a priority is given to reading by column or wavelength It is most important to understand this priority with multi wavelength readings In column priority an instrument will read the entire microplate or chosen number of strips at the first wavelength and will then go back and read the microplate at the second and subsequent wavelengths 4 44 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments SoftMax Pr
355. from one group can relate to or contain infor mation derived from columns in another group You can also create sum maries which consist of formulas and text e Notes sections allow you to enter summaries which consist of formulas and related text Use of formulas in SoftMax Pro is described in a separate manual The SoftMax Pro Formula Guide SoftMax Pro User s Manual Chapter 9 File Management and Printing File Creation and Management lees es 9 3 Default PrOtOcOh ween 1 text 8 reo Phe TEE e EDS 9 3 Locating Protocol and Data Files 0000 9 5 Crea ne New Tilger ociste way Soe PERSE ERES 9 5 Openi Filesi PCT ERUUTETT 9 6 bavine Tales Mantal errate er sur egw seh wera rer P 9 8 PANOSAVe eI 9 9 PUNE reuroriem s MES Tw E SEN PP cee io eae ee 9 11 Th Printed ReDpOP suec nou odd obe hae oe rere uid 9 12 Customizing the Repottbozs ovv a irre tT ERU CE TE 9 12 The Print Dialoe BOX ssrass vide xdi open ege 9 14 Exporting Importing Information 0000s eee eee 9 16 Exporting Data 5 0 24 2 541 9 540 eee 37998 Gta a eoe d Od 9 16 Importing Data from Analyst Acquest ScreenStation anc EBIPRS vive Sousa aortas ees aU edidi s eres 9 21 Importing and Exporting Templates suus 9 24 EXpOrtine Graph seare T ens letend t eaten d edad eb ets 9 25 COpy TOP and Paste aset oer OU MERE ayer wee PEE 9 25 File DEOIeCBON ovp ed ase VOR WE pti acd baee e edd 9 29 Password Protection
356. g as well as add page numbers to the report which are printed at the bottom right corner of each page These are all enabled by default until you disable them by clicking in the box with the checkmark next to each option which will deselect that option If a Notes section is longer than can be printed on one page a line may be cut off not printed at the bottom of the first page To fix this problem insert a carriage return in the appropriate place above the page break in the Notes section and print again Changing the Section Order When sections are created either automatically by SoftMax Pro or by you their order may not be optimal for either viewing or printing The order of sections in the window determines how they will print in the report and you may find that changing the order of sections makes it easier to view the data When you create a new section it is positioned directly after the currently active section To change the order of a section first make it active by clicking on it then click in its tool bar and drag it to a new position on top of the sec tion you want it to precede Sections may be open or closed during this pro cedure v E Standards Hah 28 ere Standards units ml v EE cuvetteset 1 E Setup kl k
357. g 384 well plates using the SpectraMax 340PC or PIus additional display options can be chosen see Figure 7 4 to make the display of data more clear Normal shows the data in standard left to right format with well A1 in the upper left corner Columns 1 through 24 are along the top and rows A through P are shown from top to bottom The wells are smaller and data is shown with fewer digits les precision Vertical the plate is shown in the same orientation as Normal except that columns 1 through 12 all rows are shown above columns 13 through 24 all rows Rotated Same as the Normal view except that it is rotated 90 degrees clock wise well A1 is in the upper right corner Large Same as the Normal view except larger This display can be seen in its entirety only if you have the page size set for legal paper 8 5 x 14 inches in landscape orientation set this in Page Setup or Preferences under the File menu In the example in Figure 7 4 the display is shown as it appears if you do not change the page size from letter size to legal Interleaved In this display the wells are shown in a format that skips every other well as follows all odd columns and rows begin in the upper left corner of the plate display and are followed by all even columns and rows So well A1 is still in the upper left corner but it is followed horizon tally by well A3 A5 A23 A2 A4 etc and vertically by well C1 E1 O1 B1 D1 etc T
358. g Wells To Read Compound Source This setting is used to describe the type of source plate that is used for com pound transfers For this tutorial choose Beckman 96 Well 2 3 mL When you are finished the dialog box should appear as shown below SoftMax Pro User s Manual 10 43 Chapter 10 Tutorials ma Instrument Settings p Beckman 96 2 3mL fa Costar 96 opaque 3mL Costar 96 Ubtm clear 3mL Costar 96 Vbtm 3mL Greiner 96 Vbtm 3mL Nume 96 VYbtm clear 3mL Polyfiltronics 96 2mL 4 Twin Troughs Beckman 20mL Trough 24 Column Plates Greiner blk clrbtm E Compound Source Figure 10 49 Instrument Settings Dialog Box Showing Compound Source 10 44 SoftMax Pro User s Manual Chapter 10 Tutorials Compound Transfer The Compound Transfer settings are used to describe the fluid transfers that will occur during the assay The settings in this portion of the dialog box work in conjunction with those in the Compound Source and Compound amp Tip Columns settings This tutorial does not make use of compound transfers Leave this set to None Triturate Trituration is a mixing step that can be set individually for fluids in the compound source plate and the assay plate Trituration is achieved by aspi rating and dispensing fluid out of and back into the same well of a plate You can set the volume of fluid to be aspirated the number of times to perform this cycle and also
359. g as defined by Lag Time described below and ending at a total num ber of readings equal to the Vmax Points setting The second and any subse quent slopes are calculated beginning at the second time point and ending at a total number of readings equal to Vmax Points The steepest slope is reported as Vmax Rate If the data plot displays fewer time points data points than the number of time points chosen for Vmax Points then all of the time points will be used to determine the slope of the data SoftMax Pro User s Manual 7 33 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 1 B 34 Table 7 1 Using Vmax Points Example Time points D aaeoa in the well data plot Number of Points Used for Data Analysis Vmax Points set to 25 25 default Vmax Points set to 5 25 Overlapping Overlapping 5 point line 5 point line segments segments SoftMax Pro will perform a linear regression on as many overlapping 5 point line segments as possible in the analysis window The slope will be determined for each line segment and then the steepest slope will be reported as the reaction rate Thus in well 2 of the example in Table 7 1 above the slope will be determined for line segments consisting of time points 1 to 5 2 to 6 3 to 7 4 to 8 5 to 9 and 6 to 10 and then the slope of the line segment with the steepest slope will be reported as the Vmax Rate The progressive slope calculations are repeated unt
360. ge limits in graph sections The data for sample blanks obtained using SoftMax will be available when these files are opened in Soft Max Pro but no calculations are performed automatically using this data Auto range limits are not available when SoftMax files are opened using SoftMax Pro so limits for the display of information in graphs must be entered manually Cross Platform Files If you have SoftMax or SoftMax Pro files that were created on a platform other than the one you are currently using you can transfer these files to your computer and use them provided you have the required software and or hardware to do so Transferring Macintosh Files to the PC L1 Third party software is available for the Windows compatible PC that allows the insertion and reading of Macintosh 3 1 2 inch diskettes if your Macin tosh is equipped with a floppy disk drive If you need to transfer files between Macintosh and PC platforms and do not have such software for the Windows compatible PC you can use PC floppy disks Zip disks or other storage media for both platforms since the Macintosh will be able to read them Macintosh computers are equipped with software that allows you to read and transfer files from PC disks Make sure that File Exchange is available in the Control Panels The process of using File Exchange is described below 1 If you have not yet enabled File Exchange on your Macintosh select Control Pan els from the Apple
361. gement and Printing Copying from Other Programs 9 26 copied contains the groups Standard and Unknown and the group Standard already exists in the destination experiment the group Unknown will be created but all information regarding the group Stan dard in the destination experiment will remain unchanged Experiments define a naming scope for the sections within them which is especially important for Group sections it allows you to have identical groups name and column format within two or more different experiments in the same file Having multiple experiments in the same file also provides you with quick access to different types of information without having to open or refer to multiple files If you are copying cuvette data and the target CuvetteSet section does not contain the appropriate cuvettes for the data that is to be pasted new cuvettes will be created automatically Please note that pasting cuvette data for only some cuvettes will clear the existing data from all other cuvettes in that sec tion For example if cuvettes A2 A4 and A6 are copied from CuvetteSet 1 and you attempt to paste this data into CuvetteSet 2 which contains cuvettes A1 through A12 the data in all twelve cuvettes will be cleared and then cuvettes A2 A4 and A6 will receive the pasted data An alert box will ask whether or not you wish to replace all the data before proceeding Plate section data CuvetteSet section data and Not
362. gement and Printing Copying to Other Programs Plate section CuvetteSet section Group table section and Notes section data can be copied from SoftMax Pro and pasted into another program that will accept the data such as a spreadsheet or word processing program No copy or paste features are available for Graph sections or Well Graphs but these graphs can be exported as either PICT or EMF files see Exporting Graphs on page 9 25 for more information Group Columns You can copy a Group column and paste it within another Group in the same experiment or into a Group of another experiment The data included in the pasted columns will be appropriate for the target group Highlight the column select Copy 3 C CTRL C from the Edit menu then acti vate the section into which you want to paste the column and select Paste 38V CTRL V from the Edit menu If you highlight a column in the destination section the column you are pasting will appear to the right of the highlighted column Otherwise it will be appended to the right of all existing columns Columns and summaries in Group sections can also be copied and pasted to other programs Data from other programs cannot be pasted into Group sec tions within SoftMax Pro however Copying Formulas and Summaries Formulas are used in Group columns custom reductions and summaries Sometimes it is useful to copy an existing formula column or summary from one area of the program to a
363. gh the list or to select multiple items by dragging across them Duplicate A command available in the Edit menu that allows you to create identical cop ies except data of all sections and experiments The name of the duplicated item will be the same as the previously active item with the word Copy appended Emission Spectral Scan Measures fluorescence or luminescence across a Spectrum of wavelengths for emitted light for a fixed excitation wavelength The default value reported for each well is the wavelength of maximum emission fluorescence or lumines cence Endpoint A single reading of optical density made at one or more wavelengths End Time Reduction parameter used to omit the end of a Kinetic Spectrum or FLEX run from data reduction Excitation Spectral Scan Measures fluorescence at a single emission wavelength across a spectrum of excitation wavelengths The default value reported for each well is the excita tion wavelength of maximum fluorescence Export Data from SoftMax Pro is copied to a new file and formatted so that it can be opened by another program such as a spreadsheet or word processing appli cation Data from some or all Plate CuvetteSet or Group sections may be exported Text exported from SoftMax Pro is tab delimited Exponential Curve Fit The exponential function used to generate this curve fit is y A B 1 exp x C More information regarding curve fits can be found in Chapters 7
364. ghlighted column or if no columns are selected after the last col umn in the Group To add a new column to the right side of the Standards Group make sure this section is active Then deselect any highlighted columns by clicking outside the column area Click the Create Column button To or choose Create Column from the Group menu A dialog box will open asking you to enter information for the new column Calculation Name Colx8 Formula Decimal Places i3 aes ues C Figure 10 72 Calculation Dialog Box 10 60 SoftMax Pro User s Manual Chapter 10 Tutorials D SoftMax Pro User s Manual In this tutorial example the new column will show the maximum value obtained for each sample Type over the default column name entering MaxValue in the box next to Name Now enter Max Values in the box next to Formula and click OK J Be sure to enter the formula exactly as shown The dialog box will close and a new column will be shown at the right of the previous columns showing the maximum value obtained for each sample Concentration Values Plean alue US Activation 0 010 826 221 Sr3 994 D ns 918918 19416z6 58597 Figure 10 73
365. gistic curve are based on the assumption that the curve has a high and a low asymptote and a certain steepness for the linear portion around the inflection point between the asymptotes If a sufficient number of points do not define the inflection point and the asymptotes you will receive a no fit error message or an inappro priate fit For best results make sure that the 4 parameter fit is appropriate for your assay and that the entire range of the assay is represented in your stan dards If your standards do not clearly define both a high and low asymptote the spline fit may be more appropriate for your assay Judging a Good Curve Fit The correlation coefficient is also known as the goodness of fit value because it shows how good a fit exists between the computer curve and the standard data points It measures the degree to which the points fall on the computer curve and may give an indication as to what residuals are to be expected The correlation coefficient is popularly known as R and is between 1 and 1 inclu sive When the data points lie on a perfectly straight line with negative slope then R 1 If the correlation coefficient is not suitably close to 1 or 1 a per fect fit you can simply apply one of the other types of curves without having to reread the plate or cuvette For nonlinear curve fits SoftMax Pro performs transformations that cause the curve to become more linear The correlation coefficients are de
366. group is selected in the Group drop down list The following illustration shows how the tool bar will appear under various circumstances Group ES mr Clea r When Plate or Template Blank is selected Group Unknowns Y cnt m ad A group is selected that does not contain sample descriptor information such as Unknowns column type Edit s le Stan Group Standards Y d i Clear Concentration b Jaam A group is selected for which a concentration has been defined such as Standards column type w L Ian A group is selected for which a dilution factor is defined such as UnknownsDilution column type Figure 4 18 The Appearance of the Template Editor Tool Bar under Different Circumstances The tool bar is divided into three areas the Group area the Sample area and the Assign area The Group Area The Group area contains the Group drop down list which allows you to assign wells to existing groups create new groups or modify using the Edit button existing groups In addition it contains the Clear button which allows you to clear well assignments in the template Group Drop Down List This drop down list consists of two areas divided by a horizontal gray line The two items below the gray line Blank and New are always available Items above the gray line are custom groups that may be created as you wish The Group drop down list will appear differently depending upon
367. gth Combination Lm1 Data Mode Absorbance Plate Blank Used L1 2 0 13 v EDI cuvettesets CuvetteSet 1 AT 7 A Kinetic ly Helen Data 716 99 9 47 AP Data 716 99 9 54 AP Data Midd Lid Li Cuvette Ref Wo Reference Ref Wo Reference Ref Mo Reference LAE mE mo 0 518 0 365 BAT 0475 oF eo M BUS Lag Time 0 00 End Time 3 00 OD Min 0 OG Plax 1 Vinax Pts 24 24 Wavelength Combination Lm1 Data Mode Absorbance Figure 7 17 Masked Wells in a Plate Section and a CuvetteSet Section To remove masking from one or more wells highlight the masked well s and click the Mask button in the Plate section tool bar toggles masking on and off or choose Mask from the Plate or CuvetteSet menu again The diagonal lines covering those wells and masking function will be removed 7 22 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Data Reduction Raw data received from the MDC Microplate System instrument can be dis played in an analyzed or reduced form The reduction process within Soft Max Pro is based on formulas that reduce the raw data to show a single number for each well or cuvette Further analysis of this reduced number then takes place in the Group and Graph sections SoftMax Pro performs calculations hierarchically when reducing the optical density information collected from the instrument Calculations in the Plate
368. h plot contains the same number of data points and has matching X axis coordinates 93831 When choosing the Connect Points option the order in which points are connected is sample name dependent SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Axes Options aH Double clicking either graph axis clicking the X axis or Y axis button in the Graph section toolbar or selecting either X axis or Y axis from the Graph menu opens a dialog box concerning the configuration of that axis Figure 7 55 shows the dialog box for the X axis X Axis Settings amp Linear Q Log Figure 7 55 X Axis Dialog Box The default settings are shown in the Figure above You can change these as desired for either or both axes Axis Settings Enabling the Auto range option causes the graph to be drawn such that all data points are shown by automatically adjusting the minimum and maxi mum range settings to encompass the data You can disable the Auto range function by clicking the checkbox or by changing either or both values for the Min and or Max settings Formulas in SoftMax Pro SoftMax Pro User s Manual You can enter custom calculations in several places within SoftMax Pro to provide the information you desire e n Plate and CuvetteSet sections you can specify formulas to be used for wavelength reduction and Kinetic Spectrum reductions n the Group table sections you can vi
369. h the computer is connected and establishes a communication rate accordingly This option allows you to view the baud rate that is being used and to manually set a different rate but this should not be done unless a serial communications problem occurs see Chapter 11 Troubleshooting for more information Export Format SoftMax Pro User s Manual Export Format Time Iw Include Labels Interpolate Wells Add Filename and Date Figure 2 10 Export Format selection in Preferences dialog box The preferences settings regarding the Export Format allow you to choose whether to export data as it appears in plate format or linearly over time The option to include text labels along with numerical data and or to add the filename and date can be activated by clicking the checkboxes next to those items The option for Interpolate Wells is available only with the FlexSta tion instrument For more information on the Export function refer to Exporting Importing Information on page 9 16 Chapter 2 Installation 2 18 Reader Filters The Reader pop up menu indicates which instrument is currently connected to the computer When the computer is connected to a MDC Microplate Sys tem instrument the instrument type shown in the box will be set to that instrument type Emax V max LI max THERMOmax VERSAmax SPECTRA max SPECTRAmax PLUS SPECTRAmax PLUS 384 SPECTRAmax 190 SPECTRAmax 340PC
370. h the reduced number Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments gt Ca aen EA max units per sec Plate 1 1 2 3 4 5 B 7 8 qg 0 WW 12 Kinetic Time 5 00 0 696 9 632 1O 635 11 773 12 967 14 267 15 713 117 372 13 255 21 376 23 602 26 192 Int i erval 0 23 Reads 14 10 53511 77312 35714 257 15 71817 372 13 255 21 375 23 502 25 132 28 332 32 037 E i i x Fluorescence Bottom read 12 967 14 267 15 71917 372119 255 21 376 23 602 26 1 92 25 992 32 097 35 442 59 207 Ex Em cCutaff Lind 485 538 530 D i5 7191737249255 24 376 23 502 26 192 28 292 32 097 35 44239 207 43 287 45 222 esee seo necare uhr e enr es i419 255 21 376 23 502 26 19228 89232 097 35 442 39 207 43 287 45 222 47 233 49 407 Automix Off Calibrate On PMT Medium 3502 26 192 28 992 32 097 35 442 39 207 43 287 45 22247 233 49 407 51 621 53 8 992 32 097 35 442 39 207 43 287 45 222 47 233 49 407 51 821 53 913 56 368 59 161 Lag Time 0 00 35 44239 207 43 287 45 222147 233 49 407 51 621 53 843 56 366 58 161 61 6365 24 7 PF Min 0 RFU Max 20000 Wavelength Combination Lin wimax Pte 14 14 Plate Last Read 4 23PM 2 5 2001 6 7 to 65 Figure 8 6 Kinetic Data Displayed as Threshold with Reduced Number Ranged When the Ranged display is chosen raw data that falls between the high a
371. haracter Set B 1 file 2 21 3 16 3 18 3 26 3 34 3 36 B 7 string based messaging 3 40 Assays folder 2 23 3 24 9 3 9 4 12 10 menu 2 23 3 24 3 32 9 3 9 4 12 10 Audit Trail 6 12 6 25 Author name B 1 AutoCalibrate 4 4 4 18 4 39 4 40 4 44 5 4 5 18 5 34 5 43 B 2 Automix 3 31 4 4 4 5 4 10 4 17 4 43 5 4 5 11 5 17 5 38 5 44 B 2 B 14 button 3 7 3 8 Autoprint 2 22 3 29 9 11 11 12 Preferences setting 2 16 with ROBOmax 9 10 SoftMax Pro User s Manual AutoRead 5 38 B 2 Autosave 2 20 2 21 3 29 9 9 9 10 Append to File 2 21 Create New File 2 21 directory 12 12 file prefix 2 21 Preferences setting 2 16 with ROBOmax 9 10 Autosize command 3 20 3 38 7 43 8 40 10 26 10 58 B Background Constant subtraction 4 15 4 19 7 23 Interference 4 15 5 14 5 15 interference 4 15 Baud B 2 Blanking 4 33 4 34 5 30 5 31 B 2 B 3 B 14 blanks in combination 4 34 5 31 CuvetteSet section B 2 group associated blanks 4 33 4 34 5 31 B 2 B 3 plate blank 4 33 5 31 7 26 10 12 11 14 B 3 pre read plate blank 3 31 4 15 4 19 4 33 5 30 7 26 7 37 B 2 Ref 4 33 B 2 template blank 4 34 B 2 Bottom Read B 3 C Cable connections 2 7 2 16 11 10 11 11 Macintosh 2 3 2 4 11 12 PC 2 5 B 10 B 13 Calculation dialog box 3 14 3 20 7 38 7 46 7 47 8 36 8 44 8 45 hierarchical 7 23 8 19 Calibration 3 31 4 44 5 11 Change Password Enterprise Administrator 6 20 Checkbox B 3 Chooser 2 4 3 27
372. hat Fit Log Logt Graphz 1 concentration y CA DMTHGUCPBME A B e D Plot 1 Grouped concentration vwa Meanvalue 0 1 1 247 11 262 0 305 Figure 8 45 Graph with Log Logit Fit Some publications show the log logit equation in another form log e bx b log c where y L4 x log A different algebraic expression of these is y EE D x C The difference between the log logit fit and the 4 parameter fit are in the way the coefficients A and D are calculated For the log logit method the standard values for the lowest and highest values of x are used The corresponding y values are assigned to A and D respectively Based on these fixed values for A and D the algorithm then computes values for B and C This technique works well if there are standard points along the upper and lower asymptotes If this is not true the log logit fit should be avoided in favor of the 4 parameter logistic In SoftMax Pro 2 x the 4 parameter logistic curve fit uses 2000 iterations If the fit does not converge in this number of iterations the program will report a fit error The curve fitting algorithm for the 4 parameter equation was based on the Levenberg Marquardt Method Discussion of this method can be found in Numerical Recipes in C The Art of Scientific Computing by William H Press Brian P Flannery Saul A Teukolski and William T Vetterling published by Cambridge University Press New York 1988 Sof
373. he Command menu If you have created more than one Plate section and no Plate section is active choosing the Read command will cause a dialog box to appear asking you to choose which section to read see Figure 5 49 Select the correct section and then click OK to close the dialog box If a Plate section is active choosing the Read command will read that plate Select Section Sections Figure 5 49 Select Section Dialog Box If you select a Plate section that already contains data and choose to Read again an alert appears asking you to confirm replacing data in the destina tion plate see Figure 5 50 If you select Repl ace the reading will proceed and the previous data will be replaced with new data 5 51 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Replace data in Plate 1 Cancel Replace Figure 5 50 Alert Regarding Replacing Data for Plate Section General Information about a Reading After beginning a reading the status bar will show the actions that are occur ring in the instrument along with a box showing the elapsed time for each action in minutes and seconds The button will change to Stop allowing you to terminate a reading if necessary An example of the appear ance of the status bar during a FLEX reading is shown below 221 E3 Pieter Reading 34 of 34 0 00 19 Figure 5 51 Status Bar during a FLEX Reading Data Display During a Reading The
374. he Group section for Standard2 kd Standard ry E r Standard pg ml Sample Concentration Wells Values _Meanvalis StdDev CV ETT RN tig TT E 420 000 420 000 0 000 Stal 0 500 410 000 410 000 0 000 00 00 Stao 0250 3 ooog 410 000 0 000 0 0 Stad 0125 C4 10 009 410 000 0 000 0 0 Stas O00ezjcS ooog 410 000 0 000 0 0 jSta amp 0 081 ce ooog 410 000 0 000 0 0 Smallest standard value 410 000 Largest standard value 420 000 Figure 7 37 Standard2 Group Section 3 Anew Graph section is created by selecting New Graph from the Experiment menu This causes the Plots dialog box to open automatically The default name for the first plot Plot 1 is then changed by typing a new name over it to Standard 1 Clicking OK opens the Graph Options dialog box which allows you to choose the graph type modify the fonts used for the graph and so on Choices should be made as follows Standard1 for the group to be plotted the concentration of Standard1 for the X axis and values of Standard1 for the Y axis No error bars are added 7 50 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Graph Options Title Graph 1 Height Type onts ud Sample Text Eh Cluster bar Sample Text Set hil J Stack bar Legend Connect points 5 le Text Plot symbols Plate pee o Standard 4A Concentration Standard Y Yales
375. he Plate menu Change Instrument Settings Allows the user to edit the settings for the instrument through Instrument Setup in the Control menu or by clicking the Setup button on the plate sec tion Change Notes Text Allows the user to modify or add to the text contained in the Notes section of an experiment Mask UnMask Wells In the template editor a user can select which wells will be excluded from analysis Read Empty Plate and Cuvette Sections Allows a user to perform a reading if a plate does not contain data Overwrite Plate and Cuvette Data Allows a user to perform a read that replaces data that is already present in a plate section Remove signatures from e statements Allows a user to remove the signatures from a statement making that experi ment editable again Modify User To modify a user select that user from the Users list box on the Users property page and then click the Modify User button The Modify User dialog box will open Remove a User To remove a user select that user from the Users list box in the Users property page and then click the Remove User button The following warning mes sage will appear asking for confirmation from the administrator to prevent accidental removal of a user SoftMax Pro User s Manual 6 13 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator 6 14 Remove User MEN x You have chosen to remove John Smith From Ehe software s use
376. he Plate section tool bar toggles masking on and off or choose Mask from the Plate menu again The diagonal lines covering those wells and masking function will be removed SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Data Reduction Raw data received from the fluorescence instruments can be displayed in an analyzed or reduced form The reduction process within SoftMax Pro is based on formulas that reduce the raw data to show a single number for each well Further analysis of this reduced number then takes place in the Group and Graph sections SoftMax Pro performs calculations hierarchically when reducing the informa tion collected from the instrument Calculations in the Plate section are per formed in the order shown below If an option has either not been selected in the Instrument Settings dialog box is not available for the instrument you are using or has not been defined in the template and or Reduction dialog box SoftMax Pro starts continues with the next listed calculation 1 Wavelength reduction 2 Kinetic or Spectrum reduction 3 Group blank subtraction SoftMax Pro User s Manual You can access the Reduction dialog box in several ways by clicking the Reduction button in the tool bar of the active plate section by choosing Reduction from the Plate menu or by clicking the Reduction button in the Well Graph display The app
377. he default setting is checked enabled Turning AutoCalibrate off allows the instrument to complete readings more quickly or to begin the first reading of a Kinetic plate faster However you should allow the instru ment to perform autocalibration of at least one plate prior to disabling this function calibration settings for the last plate which included an autocalibra tion are maintained in the instrument memory NVRAM until another auto calibration is performed 4 16 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments AutoRead This feature enables the automatic reading of subsequent Plate sections in the order in which they appear within an experiment You can set intervals delay time between the plate readings if desired Blanking Pre Read Plate SoftMax Pro User s Manual The blanking selection found in the Instrument Settings dialog box is known as pre read plate blanking but pre reading the plate is only one of five types of blanking available in SoftMax Pro Understanding the differences between the types of blanking functions will enable you to choose the type of blanking that is appropriate for your assay Note that blanks can be used in combina tion and have cumulative effects Pre read plate blanking is discussed here for a description of the other types of blanking that are available and how these blanks work in combination see Blanking on page 4 33 If pre
378. he new column W Calculation X hlarme Colg Formula Decimal Places Aree Cancel Figure 10 33 Calculation Dialog Box SoftMax Pro User s Manual Chapter 10 Tutorials In this tutorial example the new column will show the maximum value obtained for each sample Type over the default column name entering MaxValue in the box next to Name Now enter Max Values in the box next to Formula and click OK J Be sure to enter the formula exactly as shown The dialog box will close and a new column will be shown at the right of the previous columns showing the maximum value obtained for each sample Figure 10 34 New MaxValue Column Appended to Group Section D View an If Statement in a Column Formula The Samples section contains a column called Outliers This column is made up of an If statement that calls upon data from another Group section to report when data points lie outside the range of the minimum and maximum Standard values An If statement in a formula defines a condition that if found to be true causes a certain result and if not true causes a different result The basic structure of an If statement is If condition resultl result2 Let s take a closer look at the individual elements that make up the Outliers formula If Values gt MinStd Standards and Values lt MaxStd Standards Outlier This statement says that if the data shown in the Values column
379. he word Outlier Looking at the Standards section you will not immediately see the MinStd or MaxStd calculations used by this If statement They are there but their names are hidden Look at the bottom of the Standards section and you will see two summaries as shown in the following figure 10 61 Chapter 10 Tutorials 10 62 Lu Ea fen E Standards paqimi Figure 10 74 Summaries in the Standards Section Double click the first summary Smallest standard value A Calculation dialog box will appear W Calculation Description Smallest standard value Formula Min Mean alue y Decimal Places Agent Cancel Figure 10 75 Calculation Dialog Box from Summary in Standards Section This dialog box contains a checkbox for Hide Name which is currently enabled Unchecking the box will allow you to view the name of the sum mary to the left of its text in the Standards section The formulas for MinStd and MaxStd are Min Mean Value Max Mean Value The MeanValue column is derived from the average of the well values found in the Values column the Standard replicates The minimum and maximum values from the MeanValue column in the Standards Group are used to create the summaries which is what the Outliers column formula uses to name its Outliers Such a formula might be useful in finding points that might be better masked not used in the calculations for example Basic information rega
380. help you get started using SoftMax Pro NOI Screens and parts of screens represented in this manual may depict either the Macintosh or the Windows version of the software The two interfaces are quite similar but show subtle differences such as where the Help menu is located etc Depending upon which com puter platform you are using which operating system you are using and the type of screen that is depicted your screen may look different from the one shown Note that Macintosh screens are depicted using MacOS versions 8 6 and later The rest of this chapter provides introductory information regarding the program and its features Chapter 2 Installation describes how to install SoftMax Pro on your computer and how to connect the computer and the instrument correctly Chapter 3 SoftMax Pro Overview explains how the SoftMax Pro soft ware program is organized and how it is used with various instruments Chapters 4 and 5 discuss how to prepare for and conduct a reading using SoftMax Pro Chapter 4 deals with absorbance instruments Chapter 5 deals with readings made using fluorescence instruments Chapter 6 discusses the Enterprise Edition of SoftMax Pro and how it works with Enterprise Administrator software Included are pre installation cau tions and installation instructions for Enterprise Administrator software along with how to set up SoftMax Pro to interface with the database created in Enterprise Administrator Chap
381. highlighted for copying the extra wells are left empty If you try to paste template information between Plate sections having differ ent plate format settings an error will result Plates must be of the same type to copy and paste template information Reading a Microplate 5 34 Fluorescence is defined as the measurement of light that is emitted after absorption or excitation from radiant activation of a compound Luminescence is the measurement of light given off by a chemical or bio chemical reaction When reading a microplate the SpectraMax Gemini fluorescence readers per form autocalibration if enabled in the Instrument Settings by reading from an internal reference chip in order to determine the appropriate scaling for RFU or RLU You can start a reading at any time after defining instrument settings It is not necessary to define groups and assign wells within the Template Editor first The values received from the instrument are raw fluorescence luminescence or time resolved fluorescence readings and are not affected by settings in the Template Editor Only instrument settings must be defined prior to reading a microplate To read a microplate open the drawer of the instrument click the drawer but ton in the status bar or choose Open Drawer from the Control menu and insert the prepared microplate Then simply click the Read button in the status bar or choose Read from the Command menu If you have created m
382. his display is most useful when the 384 well plate is composed of 4 daughter plates of 96 wells each 9 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments TU JA Raw data in 1000 s Plate 1 Normal Display 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 im gt C3 Piate 2 Raw data in 1000 s Plate 1 1 2 3 6 7 2 755 3 055 Endpoint 3 355 2 655 Fluorescence 3 955 4 355 Ex Em Cutoff Lmi 355 612 610 Auto Cutoff 4 755 5 255 5 755 6 355 Automix Off 6 955 7 655 Calibrate On PMT Auto moz inrnxc rowWnnm 8 455 9 355 Reads well 6 Endpoint Lmi Automix Off Last Read 10 253 1 595 Ex 355 Calibrate On 7 14 99 5 755 6 355 6 955 7 655 8 455 9 255 10 253 11 255 12 455 13 755 15 155 Plate Last Read Em 612 PMT Auto 7 99 AM 7 59 AM 7 14 99 Cutoff 610 Reads Vell 6 6 955 7 655 8 455 9 355 10 255 11 355 12 455 12 755 15 155 16 755 18 455 eads well 1 T 1 8 455 9 355 10 255 11 355 12 455 12 755 15 155 16 755 18 455 20 355 22 455 Auto Cutoff T OO mo m oO 60 O cx Fluorescence T T Wavelength Combination Lm1 10 255 11 255 12 455 13 755 15 155 16 755 18 455 20 255 22 455 24 855 27 455 lt 12 455 13 755 15 155 16 755 18 455 20 255 22 455 24 855 27 4553 30 255
383. his folder as the default The Assays menu will also include sub menus for all subdirectories in the default directory that contain protocols Subdirectories are only shown one level deep A subdirectory that contains other subdirecto ries will not be translated into a cascade of sub menus Only the first subdirectory name will appear in the Assays menu Opening Other Protocol Files The bottom half of the Assays menu provides a shortcut for opening other protocol files contained in the set folder These usually consist of protocols you or others create for repeated use To choose a different protocol file click the protocol file you wish to use An untitled copy of that protocol file will open in a new window SoftMax Pro User s Manual Chapter 9 File Management and Printing Locating Protocol and Data Files You may find it useful to create separate folders subdirectories to store the data files that SoftMax Pro creates In addition you may want to create and save various protocol files for use as templates for later readings In a multi user environment it might be helpful to create a folder subdirectory for each SoftMax Pro user or to create separate folders subdirectories for different types of experiments We recommend that you consider the way in which you will organize your files before creating too many of them otherwise you may find that locating a particular file among the many that fill your hard d
384. his manual has been prepared with every precaution to ensure accuracy Molecular Devices Corporation assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information This manual does not represent a commitment on the part of Molecular Devices Corporation The software described in this document is furnished under a license agreement or nondisclosure agreement The software may be used or copied only in accordance with the terms of the agreement It is against the law to copy the software on any medium except as specifically allowed in the license or nondisclosure agreement li SOFTmax PRO User s Manual SOFTMAX PRO LICENSE AGREEMENT AND WARRANTY Important Please read this license agreement carefully before opening the software package The right to use this Molecular Devices Corporation MDC software is sold only on the condition that the user agrees to the terms of this license agreement If you do not agree to the terms of this license you may return the unopened package to MDC or its authorized agent for a full refund However opening the software pack age indicates your acceptance of these terms and conditions LICENSE AGREEMENT In consideration of payment of the license fee which is part of the purchase price you paid for this product Molecular Devices Corporation MDC the licensor hereby grants a non exclusive license to you the Lic ensee User to use SoftMax
385. hz Concentration OS D ex AX BD A B Plaot 1 Standards concentration wa Means alue 0 415 434 2 Platz3 Standards Concentration ve Means alue 52 0 4334 O Plot 2 Standards Concentration vs Mean s alue 0 925 0 434 Figure 7 40 Graph Displaying Three Standards Plots You can add as many plots to a graph as you wish and these can be of the same or different types of groups If you create a graph and later wish to add one or more plots to it simply click the Graph Options button on the Graph section tool bar and then click the New button This will reopen the Plots dialog box allowing you to add or modify the plot information 7 52 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Error Bars When data is displayed as a scatter or bar graph you can choose to display error bars for the plot of the data for the X and or Y axis Error bars are lines that extend beyond a plotted value in either or both directions and graphi cally represent some amount of error in plotted data v 7 Graph s Fit s E Graph 2 0 93 0 83 0 73 0 63 0 53 0 43 1 Concentration yz amp D I 14 x C B D A Plot 1 Standards1 Concentration vs MeanYalue 0 416 Plot23 Standards3 Concentration vs Mean alue 0 62 oO Plot 2 Standards2 Concentration vs Mean alue 0 925 Figure 7 41 Plot Showing Error Bars Error Bars Choices for displaying
386. ible To see more data yet still display absolute values increase the limits for MinOD and or MaxOD found in the Reduction dialog box Figure 7 24 SoftMax Pro User s Manual 7 31 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Reduction Wavelength Combination OD Limits Dod Data Mode D e Absorbance zn 9 Transmittance e Absolute Values one J Figure 7 24 Limits in Reduction Dialog Box If you increase the MaxOD setting to be at least as large as the largest absor bance value expected most or all of the data plots should appear in the data display In the example shown in Figure 7 23 if the MaxOD value is increased from 0 5 to 2 0 the plots then appear as shown in Figure 7 25 v E3 Plate F4 Plate 1 Kinetic Time 3 Lu Interval 0 24 13 amp utoamix Off Calibrate On Lag Time 0 00 End Time 5 00 r m m FF AA M F Ymax Pts 13713 Wavelength Combination Lm1 Data Mode Absorbance Flate Blank Used Flate Last Fead 11 54 AM 7716799 Figure 7 25 Kinetic Reduction with Absolute Values Enabled and MaxOD Value Increased 7 32 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Kinetic Reduction Kinetic reductions are applied to the list of numbers the value at each time point after the wavelength combination formula is applied Vmax per min and Vmax per sec reductions are available f
387. ick Print or OK Printer General 8 24 Print Copies LI Pages amp All From HE To EE Cancel p Puper SOHUFPEB oo oreste gue Destination DA sans amp AI QFirstfrom Auto Select amp Printer Q PDF IN Experinenttige Mf Date and Time Mf File Name Lf Page Numbers Print All i Print Report Figure 10 41 Print Dialog Box for Macintosh Print Ki E Printer Name Epson Stylus 800 ESC P 2 Properties Status Ready Type Epson Stylus 800 ESC P 2 Where LPT1 Comment Print to file m Print range m Copies C All 9999 pages Number of copies fi f Pages from 1 to 12 iu 2 jJ Collate m Options v Experiment Title v Date and Time v File Name v Page Numbers C Print All Print Report Header T ext OK Cancel Figure 10 42 Print Dialog Box for Windows You have completed this tutorial but SoftMax Pro has many other features that you will find useful The next chapter describes in detail items only briefly discussed up to this point and will provide information regarding customiz ing and using the rest of the features of SoftMax Pro 10 34 SoftMax Pro User s Manual Chapter 10 Tutorials Tutorial 2 EC 50 Assay Tutorial FLEX mode only This tutorial section will lead you through the steps required to create and run an EC50 assay utilizing a graph of concentration vs RFU This tu
388. ick in the box and type a new value using the keyboard Acceptable ranges are from 0 1 to 1000 seconds The minimum interval for FLEX mode is dependent on the Com pound Transfer settings and the number of wells to be read per column Timing Run Time secs Interval secs EN Mumber of Reads 4 linimum Interval 1 5 secs Hinimum Run Time 34 secs Figure 5 42 FLEX Timing Settings Box When you have made the choices above the total number of readings that will result is shown below the run time If you set the interval time to be shorter than the instrument s minimum read interval a warning message will appear requiring that you choose a different interval Automix The Automix function is a patented feature that allows you to set automated shaking of the microplate The options available for Automix depend on the read mode chosen and the type of instrument connected or chosen in the Preferences SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Automix e amp utomix z Automix i Li Before First Read J i Li Before First Read D Between Reads Figure 5 43 Automix Settings Box Before First Read Click the checkbox next to Before First Read to shake the microplate before the first reading Enabling Automix will shake the plate for the set amount of time before the first wavelength reading only You can accept the value shown for the amount of time to shake th
389. ick the name of the Notes section Introduction to the right of the Notes section icon New Window Opens the active section in a separate window Mouse Equivalent double click the icon in the Notes section Graph Menu This menu item changes to show CuvetteSet Plate Notes Graph or Group Graph Options dependin gon the type of section that is currently active If no section is active X Axis this menu item does not appear Axis Graph Options You can select from three types of graphs Scatter with Export Graph symbols plotted and or the points connected Cluster Bar or Stack Bar and you can set the height of the graph to be something other than the default percentage 10075 The font used for the title legend and or axes can be changed and or modified in size and style Plots can be added deleted or edited Editing plots allows you to change the plot name its source from which experiment and group as well as the items to be plotted for example concentration vs values Button Equivalent in the Graph section Section Name New Window X axis Sets the X axis parameters tick marks scale label gridlines and autorange Button Equivalent in the Graph section Y axis Sets the Y axis parameters tick marks scale label gridlines and autorange Button Equivalent in the Graph section Section Name Allows you to change the name of the Graph section Mouse Equivalent
390. idth boundary If this occurs the columns will disappear off the edge of the screen but are really still there To bring these columns back into view reduce the width of the columns that are still visible until the others reappear 8 39 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments l k 8 40 Divider being dragged to the left Standards g ml Sample Concentration wellis Values Meanvalue Std Deje 0 250 C3 C4 Figure 8 26 Dragging a Column Divider Line If the width of a section extends beyond the page width boundary the portion that is not visible will not be printed To size two or more columns to the same width highlight those columns and then drag one of the dividing lines to the desired width The width of all high lighted columns will be sized the same as the column to the left of the divid ing line you are moving Columns can also be resized to the minimum amount of space required by choosing Autosize from the Group menu By highlighting the entire table or individual columns and then choosing Autosize the amount of space each column requires will be reduced to the minimum size required by the largest piece of text in that column Double clicking the line dividing two columns causes the single column to the left of the divider to be sized according to the largest amount of text in that column Showing and Editing Existing Column Formulas When group se
391. igure 3 7 Figure 3 8 Figure 3 9 Figure 3 10 Figure 3 11 Figure 3 12 Figure 3 13 Figure 3 14 Figure 3 15 Figure 3 16 Figure 4 1 Figure 4 2 Figure 4 3 Figure 4 4 Figure 4 5 Figure 4 6 Figure 4 7 Figure 4 8 Figure 4 9 Help Menu for SoftMax Pro for the Macintosh 0002 cece eee eee 1 9 Form la Help WmGOW e voa ttai CE EHI hyd ME PP bess RETO paie ads 1 9 Help Menu for SoftMax Pro for Windows 0 0 ce eee eee eee eee 1 10 Help Contents for SoftMax Pro for Windows eeleeeeeeee eee 1 10 Using the Search Function within Help in SoftMax Pro for Windows 1 11 Choose a Topic within Help in SoftMax Pro for Windows s sss 1 11 Hypertext Links within SoftMax Pro for Windows ssesssssss 1 12 Serial Cables for Macintosh lssseeeeeeeeeeeeeeee eres 2 3 Serial Cables for USB Macintosh 2 cece eee eee eese 2 4 Seria Caples O00 a D E eestor shea Eger aes Seemed 2 5 Registration Dialog Box 0 ee cc cence eens 2 13 SoftMax Pro Title Screen Examples isc dscns deem nem d ees 2 14 SoftMax Pro Default Protocol Untitled Window esee esses 2 14 Preferences Dialog Box Windows top Macintosh bottom 2 15 Serial Port Selection in Preferences Dialog Box 0 00 eee eee eee 2 16 Serial Port Speed Selection in Preferences Dialog Box FlexStation only 2 17 Export Format selection in Prefere
392. igure 8 6 Figure 8 7 Figure 8 8 Figure 8 9 Figure 8 10 Figure 8 11 Figure 8 12 Figure 8 13 Figure 8 14 Figure 8 15 Figure 8 16 Figure 8 17 Figure 8 18 Figure 8 19 Figure 8 20 Figure 8 21 Figure 8 22 Figure 8 23 Figure 8 24 Figure 8 25 Formula Button Activated and Column Formulas Shown Column Formula Dialog BOX errore bhe temere it nEn eE EEEE TU ees Calculation Didlo DOX2a dud ewe es pa reia edad dd dd qus 9 dnd ac qd Graph Options Dialog BOX iacit t Rare Se iq da bun ad brad ge Standards and Unknowns Groups Created llslleleleeel eese bLandard2 Group SSCHOBos oua cns gn tr arai he ot wp SER UR RU va E RR ed oes Graph Options Dialog Box with Plot Name Changed to Standard 1 Graph Options Dialog Box with Second Plot Added for Standard 2 Graph Displaying Three Standards Plots Plot Show ie ESOFDAISL d cuoc ssc cadant ded meurt s Ui dag ser iare a rares Graph wili NOBIE eS equ Pra poa T uoa dd haud qd Dada E E Gong MERECE Graph with Semi Log Fit Graph with Log Log Fit Graph with Quadratic EIE ouea ao aurae hd d erx quus wq EpL Ha RO denn qs ed Graph with 4 Parameter EIE 222 ade sep erp RE Ea due Eras PR PACEM RR estes Graph with Eog Eoglt Eit ooa edt pe pRta pesa eser S obe p edidi eid Graph with Point to Point Fit Graph with Cubic Spline Fit Graph with Exponential Fit Graph Options Dialog BOX siis tpe E eura eeLetURe HEU uq qas mE SA Pas Scatter
393. il the last included data point is reached as defined by the End Time or MaxOD see below The maximum positive or negative slope is reported as Vmax mOD min Vmax Points can be set from 2 to the total number of points in the curve The num ber of Vmax Points can be set before the plate is read and can be changed after the plate is read The data display will update automatically each time The Vmax Points setting does not affect data collection The height of the Y axis in the raw data plots or plate display is con trolled using MinOD and MaxOD The default settings are MinOD 0 MaxOD 1 0 The width of the X axis in the raw data plots or plate display is con trolled using Lag Time and End Time If either the MaxOD or End Time setting excludes points such that a total number of readings equal to Vmax Points cannot be used then the maximum included points will be used Time to Vmax This is an alternative method for analyzing non linear Kinetic reactions which reports the elapsed time until the maximum reaction rate is reached rather than reporting the maximum rate itself Used in conjunction with Vmax Points Time to Vmax is the time to the midpoint of the line defined by Vmax Points and used to calculate Vmax This elapsed time data is useful for appli cations including coagulation chemistry where the changing concentration of the reagents does not change Vmax but rather will change the time at which the reaction reaches the
394. in both the Template Editor for a Plate section and a CuvetteSet section however the result of clearing wells or cuvettes differs after you close the Template Editor and return to the Plate section or CuvetteSet section data dis play In a Plate section data for cleared wells will no longer be shown in the data display In a CuvetteSet section however data for cuvettes that have been cleared in the Template Editor will still be shown for both reading and reference The Sample Area The Sample area contains the Sample drop down list and sample descriptor values active if defined in the Group Settings dialog box or if assigned auto matically by SoftMax Pro Sample 15ta01 Sample Name or ID Concentration h a egi Sample Descriptor Figure 4 23 Modifying Sample Names and Descriptor Values in the Template Editor NISSENGNR When selecting wells or cuvettes to change sample names or descriptors be sure all wells or cuvettes are replicates of the same sample and group or that you want them to become replicates of the same sample and group SoftMax Pro does not prohibit selecting individual wells cuvettes from different samples and or groups for example and changing their descriptions and they will change to become the same Sample Drop Down List The sample drop down list consists of two areas divided by a horizontal gray line Before other sample names have been defined a single pre defined name BL appears be
395. incidental or consequential damages so the above limitations or exclusions may not apply to you SOFTmax PRO User s Manual lil CLARIFICATION Life Science Edition SoftMax Pro for Life Sciences is licenced to one MDC microplate reader system Simultaneous use of the software with more than one microplate reader is expressly prohibited Simultaneous use of the software on three 3 additional computers in addition to the computer controlling the instrument if applicable solely for the purpose of analyzing previously acquired data is expressly permited Drug Discovery Edition SoftMax Pro for Drug Discovery is licenced to one MDC microplate reader system Simultaneous use of the software with more than one microplate reader is expressly prohibited Simultaneous use of the software on three 3 additional computers in addition to the computer controlling the instrument if applicable solely for the purpose of analyzing previously acquired data is expressly permited Enterprise Edition SoftMax Pro Enterprise is sold as a site license A license for each reader connected computer and analysis seat computer needs to be purchased Additional software licenses can be purchased by calling our Customer Service department at 800 635 53577 DURATION AND TERMINATION The License is effective from the date of the first use of the Software by the User until terminated or until the expiration of fifty years whichever comes first The User m
396. ing the Copy 3 C CTRL C com mand all plate data will be copied and pasting will replace all data in the destination plate PathCheck data is not copied It is possible however to copy individual cuvettes or groups of cuvettes and paste these into other CuvetteSet sections If the Instrument Settings of the target section do not match the settings from the section being copied the Instrument Settings for the target section will be updated appropriately to match Templates can also be copied from one Plate section to another or from one CuvetteSet section to another within SoftMax Pro However the effects of this action depend on whether you are pasting the template to another section in the same experiment from which it was copied or to a section in a different experiment Pasting a template to a section in the same experiment from which it was copied is useful for cross plate analyses since corresponding wells in each plate will be assigned to the same group and sample Pasting a template to a section in a different experiment will create new groups in that experiment if those groups have not previously been created in that experiment If you are pasting templates between experiments and groups from the source template do not exist in the destination experiment pasting a template will create them Existing groups of the same name will not be changed by the pasting process For example if the template being 9 25 Chapter 9 File Mana
397. ings 5 PMT Auto B i Automix Before Off Assay Plate Type 96 Well Standard Wells To Read Read entire plate Cancel Read Type Fluorescence is the default and is correct for this tutorial example Wavelengths 1 Excitation 485 Emission 538 Auto Cutoff enabled Sensitivity Readings 6 default PMT Auto default Automix Automix Before Off default AutoCalibrate On default Assay Plate Type 96 Well Standard default Wells to Read Read entire plate default AutoRead Off default 10 9 Chapter 10 Tutorials 3 Read Type Fluorescence instruments only Choices are fluorescence luminescence or time resolved fluorescence The default setting fluorescence is used in this tutorial so no change is necessary Wavelengths Click the word Wavelengths on the left side of the Instrument Settings dia log box to make it the active tab A black bar will appear over the word and the right side of this dialog box will then show the specific settings for the wavelengths selection If the settings are not correct as shown click the arrow next to the number of wavelengths and choose 1 from the drop down list then click the arrow next to the wavelength shown and choose the appropri ate value s for your instrument see Table 10 1 on page 10 9 The figure below shows what the Instrument Settings dialog
398. ings dialog box which can be accessed in three ways 1 Choose Instrument Setup from the Control menu 2 Click the Setup button in the tool bar of the active expanded Plate or CuvetteSet section or 3 Double click anywhere in the settings box located to the right of the well grid in the active expanded Plate or CuvetteSet section You may create more than one Plate or CuvetteSet section in a single experiment and the instrument settings associated with each Plate CuvetteSet section can be different Instrument Setup can only be chosen while a Plate CuvetteSet section is active and the set tings you choose will affect only that section A new Plate or Cuvette Set section that is created in an experiment that contains an active Plate or CuvetteSet section will contain the same instrument settings found in the previously active section The settings for the new sec tion can be modified as desired 4 4 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Table 4 1 lists all possible instrument settings choices based on instrument type When you have defined the instrument settings click the button to close the Instrument Settings dialog box After closing a summary of the instrument settings will appear in the box to the right of the data display in the Plate CuvetteSet section The MDC Microplate System instrument will use these settings to read the plate or cuvette
399. inutes Well Scan Editor Pattern Fill Density 7 Plate Type 24 Vell Costar Strips Read entire plate Cancel Figure A 1 Well Scan Editor option In the Display dialog for Well Scan plates the user can choose between a gray scale display showing reduced values or showing both With multiple wave length pair Well Scans the user can choose between viewing the data from different wavelengths separately or together The default reduction is the average of all points in a scan The minimum value maximum value and Custom reductions are included as in the reduction drop down list Plate Sections default to presenting the points read in a Well Scan in gray scale For multiple wavelength pair Well Scans the readings from different wavelength pairs are shown in different colors The first wavelength pair is shown in black The second wavelength pair is shown in blue The third wavelength pair is shown in read The fourth wavelength pair is shown in yellow For each position in a Well Scan the color for the wavelength pair with the highest reading is shown Well scan data can also be viewed in well graphs The SoftMax Pro export format has been extended to include Well Scans Well scan has been added to the read mode field field 5 The number of points read is reported in field 9 The Well Scan pattern is reported in field 10 The Well Scan density is reported in field 11 The data from each position is output in
400. ired from the instrument and define how the data will be dis played and used in reduction CuvetteSet sections can display samples in three different ways three samples per row one sample per row and in a grid of cells which corresponds to a microplate format B 4 SoftMax Pro User s Manual Appendix B Glossary of Terms Cut A function common to most software applications that removes a selection of text or graphics and places it on the clipboard The cut function is enabled by pressing 3 X on the Macintosh and CTRL X in Windows Cut and Paste A method of moving selected text or graphics from one location to a different location in the same file or into another file which could be in an application other than SoftMax Pro Data Display Located in the Plate section or CuvetteSet section the data display is a grid that corresponds to the layout of the wells in the microplate for specialized cuvette displays see CuvetteSet Section above This display can show the results of the reading in various formats for example raw signal data OD RFU RLU raw 76 Iransmittance for absorbance instruments only reduced data number or plot Threshold Ranged and Gray Scale Data File Contains the raw data collected from the instrument as well as any associated sections or settings Notes Graphs Groups Instrument Setup Display Options and Data Reductions Data Mode Reduction option to view absorbance data as OD abso
401. is not correct you may change it here If you did not already do so specify the starting value for the first sample in the series Values for subsequent samples are calculated from this starting value Choose the operator for the series or from the Step by drop down list Enter the starting value for the series in the Starting value box The increment is entered in the box to the right of the Step by box and below the Starting value box Figure 5 28 shows a starting value of 5 with the operator being division and the increment being 2 Thus the series will start with a value of 5 and divide each subsequent sample by 2 to produce the following series values 5 25 125 etc You must assign a fill direction from the top bottom left or right to describe how the series will fill the wells and a number of replicates Various options are diagrammed in Figure 5 29 using a 4 x4 block of wells for more information about replicates see Replicate Samples and Samples in a Series on page 5 27 The maximum number of repli cates depends on the selection of wells if you are filling down from the top for example and you have chosen a block of wells that is 4 wells wide by 8 wells high the maximum number of replicates will be four with the same block of wells selected filling from the left would allow eight replicates If the number of replicates you choose does not divide evenly into the number of rows o
402. isk difficult and or time consuming Creating New Files SoftMax Pro User s Manual Selecting New 3 N CTRL N from the File menu causes a new untitled file to open creating a new window on the screen The initial contents of this win dow are based on the default protocol file chosen in the Preferences dialog box You can create and or open more than one SoftMax Pro file at a time Subsequently opened files will appear in separate windows in a cascading diagonal order New files can be saved as protocol files at any time either before or after data is acquired To create a new protocol file enter all the instrument settings for the reading and then choose Save or Save as from the File menu The dialog box that appears presents the option of saving a file as either a protocol or a data file the default On the Macintosh choose SoftMax Pro Protocol from the choices in the drop down menu next to Format and enter a name for the file in the text box type over Untitled In Windows choose the appro priate format from the list in the box under the heading Save File as Type Also remember to choose the folder or subdirectory in which to save this file When you have entered all the information click or OK SoftMax Pro limits the number of characters that can be used in a file name On the Macintosh file names are limited to 31 characters on the PC the limit is 59 characters for the actual name of the fi
403. ize 3 6 3 12 sizing 7 21 8 17 Windows compatible computer requirements 2 7 X X axis command 3 37 7 67 8 64 1 8 SoftMax Pro User s Manual
404. k and individual samples within this group are named BL The group associated blank wells shown in the upper right are not a separate group the samples also named BL are contained within the boundaries of the large group called Unknowns and are the same color as that group In order to save changes you have made to the template in the Win dows version of the Template Editor be sure to click the button to close the window rather than clicking the Control Menu box Click ing the Control Menu box located in the upper left of the window performs the same function as the button the window will close but any changes you have made to the template will not be saved Wu Copying and Pasting Templates Copying Complete Template Information Two template actions Copy and Paste can only be activated from the Plate or CuvetteSet menu when a Plate section or CuvetteSet section is active rather than from the Template Editor If you have created a template in a Plate section or CuvetteSet section and wish to use it in another Plate section or CuvetteSet section you may copy that template and paste it into the new sec tion The results of copying and pasting a template depend upon whether the action is performed within the same experiment or between experiment sec tions Copying and Pasting within the Same Experiment Section SoftMax Pro User s Manual To copy a template make the section containing that template ac
405. k like the one below mw Group Settings Mame Sam widil lw Sample Descriptor ECTETUR EDCOO LCD EEOED Units Juin v Figure 10 13 Samples with Dilution Added When you click the button to close the dialog box the template should look similar to the one below m Expenment 1 PlateiH Samoe san ello passion Group Sam weil Ww Series lear Dilution Factor HO fugim 12 Ci atat 3 T pom LI Nes R a PM E Steere Print Cancel LK Figure 10 14 Samples with Dilution Added The samples with dilution need to be changed to be part of a series however since we really want to include six different samples with replicates Highlight wells A11 through G12 again and click the Series button 10 17 Chapter 10 Tutorials Leave the default direction from the top down and leave the default operator of divided by 1 Enter 10 for the starting dilution The Series dialog box should look like this ms Series Start Fron First Sample v Top Msami Descriptor mmm 5 C Bottom Dilution Factor Units mE i6 Lett Starting value sep Right Step by Replicates Cancel Figure 10 15 Series Dialog Box for Samples with Dilution Clicking in the Series dialog box causes the template to look like this m Expermentit1 Plate 1 Group Em saree S rises Clear 1 2 3 4 5 B go B q 10 11 12 Print Cancel LK Figure 10 16 Samples wi
406. kly or to begin the first reading of a plate faster However you should allow the instrument to perform autocali bration of at least one plate prior to disabling this function calibration set tings for the last plate which included an autocalibration are maintained in the instrument memory NVRAM until another autocalibration is per formed Wells to Read You may choose to read only certain wells or groups of wells in the micro plate or to read the entire plate Partial plate reading is available for all read modes The time required for a number of different types of readings may be significantly reduced using this setting since the instrument does not have to read the entire plate To configure the settings for a partial plate read choose the Wells to Read setting SoftMax Pro User s Manual 5 43 Chapter 5 Reading Using Fluorescence or Luminescence Instruments 5 44 Highlight the wells to be read Wells must be contiguous but do not need to start with strip 1 and do not need to be complete rows or columns and a single well may be chosen if desired If you enable a partial plate reading only the wells to be read will be visible in the data display indicating that no data will be collected for the other wells all wells will be present in the Tem plate Editor however Timing Enter the total run time and the time interval between readings A default value will be shown for the run time to change this value cl
407. l Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator Figure 6 1 Administrator s Dialog Box Users Tab shown Options available from this dialog box are described below Users Page Password Aging The administrator may enable Password Aging by specify ing the duration of the password expiration period months Once enabled each user s password will expire after the specified duration Each user will be warned of the expiration 15 days prior to the actual expiration date Idle Time out The administrator may enable the Idle Timeout by specifying a time in minutes When enabled the software program will lock itself after the specified period of inactivity and the user will have to log in again in order to use the software Lock software after three logon attempts When this option is enabled if a user enters the wrong password three times in succession the software will lock A message will appear explaining that the program is locked and that the administrator must enable the path to the database again Template Page Enterprise Administrator o 1 0 Molecular Devices Corporation Users Templates Report License A template is a predefined set of permissions for Molecular Devices software An administrator can use templates to repeatedly assign a set of permissions to multiple users Show Templates for Software SoftMax Pro Defined Templates Add Template
408. l to exit the installer and close any programs and or disable virus protection and then start the installer again from step 3 above To continue with installation click Next The screen named Choose Destination Location asks you to select the drive where the software will be installed A default location will be shown on the screen If you have more than one hard drive in your computer you may change the destination to the disk drive where you wish Enterprise Administrator to reside or accept the default location Once the software has been installed the administrator must create a data base and add user information The Database Name text box displays the name of the database to which the administrator last logged on successfully If the database has been moved to another location click the button to browse for it After entering the Admin User ID and Admin Password click the button to log in The Administrator s dialog box will open providing the options to make all additions or changes to the database Enterprise Administrator o 1 0 Molecular Devices Corporation Users Templates Report License Use the list below to add or remove users and modify global options Show users for All Charles Lexington clexington Idle Time out D Minutes r Global Options z Add User Password Aging D Months Lock software after logon failures SoftMax Pro User s Manua
409. l bar that appears below the title bar at the top of the window Minimum Reduction for a Spectrum scan that reports the minimum signal within the reduction parameters SoftMax Pro User s Manual B 9 Appendix B Glossary of Terms MinOD or MinRFU or MinRLU The limit for the minimum value you wish to use for display and analysis of Kinetic or Spectrum scan data Any values that are under this limit will not be shown and will be excluded from data reduction The default is 0 To display negative Kinetics the value should be set below 0 zero Minimize Button Windows He The small box containing a down arrow at the right of the title bar Mouse users can click the Minimize button to reduce a window to its icon Keyboard users can use the Minimize command on the Control menu A minimized application continues running and you can select the icon to make it the active application Modem Port Macintosh A serial communication port on some Macintosh models that is marked with the icon of a telephone handset Normal Display The default display for 384 well plates in the Plate section NVRAM Refers to the Non Volatile Random Access Memory of the instrument Infor mation stored in the NVRAM is not lost when power to the instrument is turned off Onset OD Onset RFU or Onset RLU The change in signal required to compute the onset time for Kinetic read ings Onset Time The time it takes for a given increas
410. late System instrument you may import display and analyze data collected from either a FLIPR or Analyst instrument Data Reduction and Plotting You can reduce the data using built in for mulas or you can define custom reduction formulas More than one reduc tion can be shown and results from different microplates and or cuvettes can be compared within the same experiment Several read modes are possible using SoftMax Pro Some may be performed in either a microplate Plate section or in a cuvette CuvetteSet section oth ers may be performed only in a microplate Which read modes are available depend upon which instrument you are using e Endpoint A single reading is made at one or more wavelengths up to six separate wavelengths can be chosen using SpectraMax instruments Default values reported are optical density OD relative fluorescence units RFU or relative luminescence units RLU depending on the instrument type e Kinetic Data is collected over time and the default display is OD RFU or RLU vs time Multiple readings are made at regular intervals which you define Default data reductions for Kinetic data are Vmax Vmax per Sec Time to Vmax or Onset Time 1 3 Chapter 1 Welcome to SoftMax Pro 1 F e Spectrum Absorbance or fluorescence data is collected across a spectrum of wavelengths and displayed as OD RFU or RLU versus wavelength The default reduction displays the wavelength that corresponds to
411. lculations however are not calculated differently by SoftMax Pro for absorbance and 96 T modes Because of this Molecular Devices rec ommends that 7o T be used only for raw OD raw OD with Pre read plate blank subtraction or PathCheck corrected raw OD readings Use caution when using T on reduced numbers or any readings that apply other calculations since the data may not accurately reflect proper calcula tions You can access the Reduction dialog box in several ways by clicking the Reduction button in the tool bar of the active plate cuvette section by choosing Reduction from the Plate CuvetteSet menu or by clicking the Reduction button in the Well Graph display The appearance of the Reduc tion dialog box will be different depending on the type of read mode you have chosen and the options you have chosen in the Instrument Settings dia log box Figures 7 18 and 7 19 show the Reduction dialog boxes for a Plate sec tion and a CuvetteSet section respectively in Endpoint Kinetic and Spectrum modes along with the default reductions for each of those modes SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Default Reductions Reduction Endpoint Ej Use plate blank a Absorbance C3 Transmittance omy TJ Reduction Kinetic Custom v ILm1 V max y V max Cunits per sec Time to Ymax Onset Time Wavelength
412. le establishes instrument setup parameters such as absorbance endpoint num ber of wavelengths etc that can be used as is or modified to suit the needs of your assay Data files are the result of running a protocol file and contain all 2 9 Chapter 2 Installation 2 10 the information collected from the instrument based on the settings from the protocol file A folder called SoftMax Pro 4 3 will be installed in the area you specified on your computer s hard drive Within that folder are a number of separate files and subfolders as shown in Table 2 1 Commonly used protocol files are grouped within subfolders that describe the type of assay e g Kinetic ELISA Protocol files are included to assist with method development Each protocol file contains predefined groups that have been created based on expectation of use with that protocol Often all you need to do is open the Template Editor and assign the groups so that what is shown there matches the layout of the plate being used for the reading Should it be necessary to create or modify groups information about doing this can be found in Chapters 4 or 5 for absorbance or fluorescence instruments respectively Certain data files are also included some of which appear within the Tutorials folder some assist with learning basic operation of SoftMax Pro and are linked to written tutorials found later in this manual others are included for you to open and continue le
413. le plus a period and 3 letter extension for a total of 63 characters Using a PC it is possible to make a file name longer than this by man ually changing it in a directory listing or by using the Save As com mand in the software A file with a name longer than this limit will no longer be able to be read by SoftMax Pro and will return an error when you attempt to open it File names that are made too long using the Save As command will show corrupted characters such as y etc for any letters that fall after the 63rd If you are creating a protocol file from a file containing data first save the file as a data file if you wish to save the data and then per form a second save as a protocol file Data is not saved with a proto col file Protocol files are stationery pad files when you choose to open a protocol file you are actually presented with a copy of the file which appears as an untitled file 9 5 Chapter 9 File Management and Printing 3 Choose the location for the new file here a Assays Enter a name for the new Protocol file here D Name Untitled Format 5oftMAX PRO Document SoftMAX PRO Protocol File type 7 drop down menu Figure 9 4 Save as Dialog Box Macintosh Save this document as ey Choose the Save in X SOFT mas PRO 1 Ey Eg ss iss location for the new file here Enter a name for the new Protocol file here File type choices Fi
414. le name Untitled oe Save as type Pro Data Files pda Cancel Figure 9 5 Save as Dialog Box Windows Opening Files You can open a SoftMax Pro file by selecting Open 3 0 CTRL O from the File menu within SoftMax Pro or by double clicking on the file name or icon when you are in the Finder Macintosh or in the File Manager Windows Note that you can open SoftMax Pro files using either platform regardless of whether they were created using Macintosh or Windows see Cross Platform Files below for more information Opening SoftMax SoftMax Pro Files Earlier Versions SoftMax Pro allows you to open files from previous versions of SoftMax Pro as well as from SoftMax From within SoftMax Pro select the Open 0 CTRL 0 command from the File menu The file will open and will function in the same way as a SoftMax Pro file If you wish you can save the file in Soft Max Pro format by selecting Save As from the File menu If you do not change the name of the file and save it to the same location a message box will appear asking whether or not you want to replace the existing SoftMax file with the new SoftMax Pro version If you do not you can either choose a different location or rename the file 9 6 SoftMax Pro User s Manual Chapter 9 File Management and Printing While SoftMax files can be opened using SoftMax Pro two items do not translate completely across the programs sample blanks and auto ran
415. lections include a standard 96 well plate and a standard 384 well plate SpectraMax Gemini XS support The SpectraMax Gemini XS was added to the Reader list in the Preferences dialog The Instrument Settings dialog for this new instrument is very similar to that for the SpectraMax Gemini A new Plate Type table was provided for this instrument The Plate Type selections include standard 96 well 384 well 48 well 24 well 12 well and 6 well plates Also the Automatic setting for PMT Sensitivity is available for Spectrum reads with this instrument When performing an excitation scan with this instrument the user can select to have no excitation filter present or to have the default excitation filter present This setting defaults to having no filter present Well Scanning Read Mode SoftMax Pro User s Manual Anew read mode is provided for a Well Scan This read mode provides the ability to take readings at more than one location within a well The Well Scan read mode is available for both the SpectraMax Gemini and SpectraMax Gemini XS instruments In the Instrument Settings dialog all the options that are included for Endpoint reads are available for Well Scans One additional option is available the Well Scan Editor shown in Figure 1 The Well Scan Editor allows the user to specify how a Well Scan is performed Four patterns are provided a horizontal line a vertical line a cross which is a combination of a horizontal and vertical
416. lick the FLEX icon button to select that mode Clicking the setting name bold heading on the left side of the Instrument Settings dialog box shows the choices available for that setting in the right pane allowing you to make changes The table below shows the settings that should be chosen for this tutorial example for the FlexStation Following the table are instructions regarding how to choose those specific settings SoftMax Pro User s Manual Chapter 10 Tutorials Table 10 2 Instrument Settings for FLEX Mode Wavelengths 5 Wavelengths Ex Em Auto Cutoff On 485 525 515 Sensitivity Readings 3 PMT High Timing m Instrument Settings 4 x deb vo Endpoint Kinetic Spectrum Well Scan FLEX Options Number ot Wavelengths i Excitation Emission V Auto Cutoff SoftMax Pro User s Manual mt Bes T Ecs T Time 200 secs Interval 2 secs Reads 101 Minimum Interval 1 28 secs Automix Before Off Between Off AutoCalibrate Once Assay Plate Type 96 Well Standard clrbtm Wells To Read Read area amp 1 H10 Compound Source Beckman 96 2 3mL Cancel Wavelengths 1 Excitation 485 Emission 525 Auto Cutoff enabled 515 Sensitivity Readings 3 PMT High default Automix Automix Before Off default Between Off default AutoCalibrate Once default Assay Plate Type 96 Well Standard clrbtm default Wells to Read A1 H10 C
417. lied to sample reading See Reference Reading on page 4 41 for more information Plate Blanks Plate section only Wells can be assigned to a Blank group in the Template Editor Unlike pre read plate blanks the average value of all the wells in a plate blank group is sub tracted from all individual well values obtained during the reading of micro plates When PathCheck is selected for the SpectraMax Plus or Plus 94 190 or 340PC the Plate Blank is subtracted after pathlength correction Plate Blank subtraction may be turned off in the Reduction dialog box so that the data may be seen with or without plate blank subtraction Plate blanks are only subtracted from wells on the same plate to which they are assigned The checkbox for Plate Blank subtraction only appears in the Reduction dialog box after data has been col lected For Kinetic and Spectrum data the blank value is averaged and subtracted on a point by point basis The example below shows a graphic representation of original and average plate blank data followed by the resulting data with average subtracted SoftMax Pro User s Manual 4 33 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 1 k Time Time Time Blank Subtracted Sample Well Average of Blank Wells Sample Well Figure 4 30 Graphic Representation of Plate Blank Subtraction Template Blanks CuvetteSet section only Similar in function to the Plate Blank used with Plate sec
418. line and a grid pattern The user also specifies a density setting which determines the number of points read in the line patterns or the maximum number of horizontal and vertical points included in the grid pattern With the grid pattern points that land too close to the edges of the well are excluded All points that land within 2 8 mm of an edge are excluded Only odd numbers of points are included in density settings The minimum number of points is 3 for all plate sizes The maximum number of points depends on the well diameter To determine the maximum number of points included in density settings 5 6 mm is subtracted from the well diameter the remainder is divided by 0 8 mm and the largest odd integer that is less than this number used A 3 Appendix A SoftMax Pro Version Specifications The Well Scan Editor provides information on the number of points to be read the point spacing and the approximate read time depending on the pat tern and density settings Point spacing is determined by subtracting 5 6 mm from the well diameter and dividing the remainder by the density The mini mum point spacing is 0 8 mm W Instrument Settings Endpoint Kinetic Spectrum Well Scan Options Ex Em Auto Cutoff On Well Scan Editor 485 538 530 Sensitivity Density Readings 5 PMT Auto Automix UN E M e i Before Off Point Spacing 1 43 mm AutoCalibrate Points Per Wiel 37 on Approximate Read Time 25 m
419. llation information for Enterprise Administrator can be found in Chapter 6 Connect the Hardware NISNEGN Turn OFF the power to both the instrument and the com puter before connecting or disconnecting any components or cables Not doing so could potentially result in damage to components in either or both machines The type of cable used to connect your computer to the MDC Microplate Sys tem instruments depends upon the particular computer and also the particu lar instrument you are connecting SpectraMax and VersaMax instruments have 8 pin DIN serial ports Emax Vmax UVmax and ThermoMax instru ments have 25 pin serial ports Most older Macintosh models have 8 pin DIN round modem ports newer Macintosh models may not have a serial port but instead have a USB port PC models vary but typically have either a 9 pin or 25 pin serial port check your particular computer to determine the type of serial connection needed If your computer does not have a serial port or existing ports are in use you may need to add a serial port through the use of an adapter C Connecting to a Macintosh 0983 If your Macintosh has a single serial port refer to Chapter 11 Trou bleshooting for information regarding connection to the MDC Microplate System instrument Serial Connection The SpectraMax VersaMax and FlexStation have an 8 pin DIN serial port and require the use of an 8 pin DIN to 8 pin DIN Macintosh straight through serial cable se
420. low the gray line the sample name BL is the name reserved by SoftMax Pro for group associated blanks The item above the gray line is the sample ready to be or currently being assigned After being assigned it will be shown below BL below the gray line SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Sari le tac Concentration O mga Figure 4 24 The Sample Drop Down List When this list first appears the only selection shown below the dividing line is BL the active sample name that is ready to be assigned appears above the line When samples are assigned to a group the default name is the first 3 letters of the group name followed by a number For a group called Standard for example the default sample names would be Sta01 Sta02 Sta03 and so on Sample names can be 300 characters long however only the first five or six characters will be displayed in the template and in the Plate section You can type a sample name directly into the box next to the arrow for the drop down list by highlighting and typing over the existing sample name If one or more wells or cuvettes were highlighted typ ing a different name will replace the sample name shown in the tem plate with the name you have typed and if the name is new will add this name to those that can be chosen in the drop down list When wells or cuvettes have been selected in the template and sample names
421. lts will be shown in the boxes You can choose a wavelength from the choices using the pull down menu or simply highlight the wavelength you would like to replace and type a new wavelength within the available range of the SpectraMax instrument over the previous one Note that the pull down menu shows only common monochromator settings VersaMax Instruments Choose whether to read at one or two wavelengths At least one wavelength must always be specified Selecting two wavelengths will cause two wave length setting boxes to appear PE NHLZ ma Tr Figure 4 8 Wavelength Settings for VersaMax Instruments The wavelengths specified previously or the defaults will be shown in the boxes You can choose a wavelength from the choices in the pull down menu or simply type any wavelength within the available range of the VersaMax instrument over an existing setting The pull down menu will show only common monochromator settings Emax Vmax UVmax ThermoMax Instruments Choose either single or dual wavelength The wavelength s must be chosen from the drop down list of values The values shown in this list are filter val ues that have been entered into the filters list under Preferences in the Edit menu this list must match the actual filters installed in the instrument Wavelengths Figure 4 9 Wavelength Settings for Emax Vmax UVmax and ThermoMax Instruments SoftMax Pro User s Manual Chapter 4 Reading Microplates an
422. lumns add a column and hide replicates Viewing Formulas To view the formulas associated with all columns in the Standards Group section hold down the CTRL Shift keys while this group is active A second row will appear beneath the column titles showing the for mulas for each column Depending on the size of the column all or only part of the formula may be shown Standards ug ml Figure 10 29 A Portion of the Standard Group Section with Formulas Shown by Pressing the CTRL Shift Keys If column formulas are long as in this tutorial example they may not be seen in their entirety in the default column size To see the complete formula for a particular column click once on a column to highlight it The formula for that column will be shown in the tool bar Tf Standards Hah E4 BackConcCalc InterpX Stdm Graphe values Standards ug ml Figure 10 30 Standards Group Section with Formula Shown for Highlighted Column BackCalcConc Click the button to modify or change a formula Resize and then Autosize a Column Position the pointer on the line dividing the columns named Concentration and BackCalcConc The pointer will change to a double headed arrow Click and drag the line dividing these col umns to the right Figure 10 31 shows these actions and the resulting wider Concentration column SoftMax Pro User s Manual Chapter 10 Tutorials Double Headed Arrow Pointer v fr Standards Hat
423. ly the most stable e An Autosaved file from the robotics run can be saved as either a text txt or SoftMax Pro data pda file This selection is made in the Preferences dia log box within SoftMax Pro choose Edit Preferences e More memory will be needed to run SoftMax Pro using remote commands Depending on the robotics used and the type of file created with SoftMax Pro a computer configured with the minimum amount of RAM see Com puter System Requirements on page 2 6 will restrict the size of the file that can be generated An experiment with more than one plate of data could adversely affect Soft Max Pro s efficiency If increasing the computer s memory is not an option 12 7 Chapter 12 SoftMax Pro Remote Command Language 1 B Molecular Devices recommends using only one plate per file and also ensuring that the computer is dedicated to use for SoftMax Pro i e is not being used for other purposes while SoftMax Pro is running Molecular Devices Position 12 8 In order to integrate robots from other manufacturers with certain instru ments from Molecular Devices Corporation or to automate the export of data from SoftMax Pro to a desired LIMS package Molecular Devices has made available in this chapter the commands enabling the remote control of Soft Max Pro Molecular Devices has tested these commands but does not provide technical support for specific integration needs Molecular Devices strongly recommend
424. m data reduction The default is 0 OD MaxOD The limit for the maximum value you wish to report Any values from the reading that are above this limit will not be shown and will be excluded from data reduction The default is 1 0 OD Start Wavelength nm Specifies the limit for the minimum wavelength setting you wish to report Any values from the reading that are under this limit will not be shown and will be excluded from data reduction End Wavelength nm Specifies the limit for the maximum wavelength setting you wish to report Any values from the reading that are above this limit will not be shown and will be excluded from data reduction NOINA The settings for the Start and End wavelength settings cannot be below the lowest wavelength or above the highest wavelength entered in the Instrument Settings for the Spectrum reading Spectrum Reduction The Spectrum reduction formula is applied to the list of numbers in each well values at each wavelength after the wavelength combination formula is applied The default reduction for Spectrum readings is Lambda at Maxi mum You can choose Lambda at Minimum Maximum Minimum or Area Under Curve instead from the pop up menu or you can choose Custom and create a different reduction formula Maximum Reports the maximum absorbance optical density or percent transmittance T within the reduction parameters of MinOD and MaxOD Minimum Reports the minimum absorbance optical
425. m and maximum values from the MeanValue column in the Standards Group are used to create the summaries which is what the Outliers column formula uses to name its Outliers Such a formula might be useful in finding points that might be better masked not used in the calculations for example Basic information regarding formulas and the information they can contain can be found in Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using For a complete discussion of writing formulas in SoftMax Pro as well as detailed examples of formulas and customized proto cols refer to the Formula Reference Guide SoftMax Pro User s Manual Chapter 10 Tutorials E Hide Replicates While the Standards section is active choose Hide Repli cates from the Group menu The display before and after hiding replicates is shown in Figure 10 37 v If stendaas LY E4 F Standards pg ml Concentratio BackCalc C Well Values MeanValue Std Dev CV 1 362 B1 0 739 0 002 0 3 1 368 B2 0 741 a LL 1 009 C1 0 0 550 0 003 0 5 Standards 7 L017 C2 o 0 801 D1 0 0 434 0 003 0 7 section mu 0 7904 D2 0 Showing 0 504 E1 0 0 276 0 001 0 506 E2 0 Replicates 0 192 F1 0 0 109 Default Q195 F2 0 110 L 0 087 G1 0 0 054 0 097 G2 0 0 017 H1 0 005 0 004 0 015 H2 smallest standard value 0 004 Largest standard value 0 739 v ffi Standard
426. mand from the Macintosh and from Windows 6 6 Printer Printer 1 Destination copies 1 LI Collated Pages amp All First page from Cassette 250 sheets Remaining from Cassette 750 sheets ILL Figure 9 10 Print Dialog Box for the Macintosh You can choose to print all of the sections regardless of whether they are included in the report by clicking the option button next to Print all at the bottom left of the Print dialog box SoftMax Pro User s Manual Chapter 9 File Management and Printing SoftMax Pro User s Manual Print Ei X Printer Mame son SOO ESCVP2 OOOO Properties Status Ready Type Epson Stylus 500 ESC P 2 Wher LPT Comment Print to file Copies Number of copies li I cess cota Options v Experiment Title W Date and Time Iv File Mame I Page Numbers Print range All 9999 pages ie Pages from li to ji e C Print All Print Report Header Text OF Cancel Figure 9 11 Print Dialog Box for Windows Epson Stylus 800 Shown If more than one printer is available to your computer make sure you select the desired printer prior to selecting one of the Print options in the File menu If you start to print and discover the wrong printer is selected choose to exit from the Print dialog box Select the correct printer using the Chooser for the Macintosh or the
427. max Pro l x plate descriptor used by this file is for another instrument 4 substitute plate descrptor has been supplied Far compatibility The change will nat be permanent until you save the File IF pou want to use this file s settings with the current instrument you should first save it to another name This error message appears if you have one type of instrument connected to the computer or chosen in the Preferences and open a file that was created using a different instrument The message alerts you to the fact that a plate descriptor for the instrument that is connected or chosen has been used instead of that saved with the file If you save the file the new descriptor will be saved instead of the old If you wish to retain the old descriptor save the file under a new name Checksum Failed Plate Section Data Wee Vy eee ee Ex Em Cutoff o p P C DP FPE LDLLILII NERIS A AFA ororo rr rrr Auto Cutott Wavelength Combination Lrm1 Trans H ps 1 v 230p 0 Tritc v 250p 0723 Automix Off E Vv z30p 20 Calibrate On Checksum Failed PRT High Reads Vell 6 Lag Time 0 End Time 300 lege This report appears in the Plate section of the FlexStation instru ment only The checksum feature checks for accuracy of data received from the FlexSta tion instrument SoftMax Pro User s Manual 11 5 Chapter 11 Troubleshooting One or nore points may have been lost during this read
428. me at Maximum Time at 1 2 Maximum Slope Area Under Curve or you can choose Custom and then create a different reduction for mula Vmax The maximum slope of the Kinetic plot of relative fluorescence luminescence units versus time The default value displayed is in relative fluorescence units RFU per second or relative luminescence units RLU per second The rate is calculated with the use of Vmax Points see below which determines the number of contiguous points over which Vmax is calculated The number of Vmax Points defaults to the maximum taken during the reading for all instru ments except the FlexStation which defaults to 5 points You can change this number in the Reduction dialog box AONA Vmax Vmax sec e 60 000 A linear regression is performed to determine the slope of the line using the equation y m X B where m is the slope of the line The reaction rate is reported in RFU RLU units sec Vmax Points This setting defines the maximum size of the line segment used to determine the slope of the line used in calculating the rate of the reaction The default is the total number of points taken in the reading with the data normalized to a starting point of 0 0 as opposed to starting with the first time point The first slope is calculated for a line drawn beginning at the first reading as defined by Lag Time described below and ending at a total number of readings equal to the Vmax Points setting The second and
429. med display options include e Number or plot displays for raw RFU relative fluorescence units or RLU relative luminescence units depending on which was chosen as a read type with or without reduced number With the SpectraMax Gemini and Gemini XS you also have the option to view raw data in 1000s e g 20 000 would be shown as 20 to make numbers in wells more eas ily visible The number format use of commas or periods in numeric notation chosen for the computer operating system in general is reflected in the way that SoftMax Pro displays data e Number Plot Threshold Ranged or Grayscale with or without reduced number e If you have chosen 384 well plates from the Instrument Settings you can display the wells in different ways Normal Vertical Rotated Large or Interleaved e When FLEX mode is selected the Reduction dialog box will show all of the settings included for Kinetic mode but with the minimum kinetic interval of less than one second and have the settings for the fluidic pipetting events With FLEX mode the Reduction dialog box also includes a section titled Display Options which includes settings for Absolute Zero Base line and Percent Increase These options are discussed in detail later in this chapter Default displays are shown automatically unless you choose a different dis play option Reduction options allow you to display the raw data received from f uores cence instruments i
430. ment 3 30 File 3 26 Graph 3 37 Group 3 38 Notes 3 36 Plate 3 33 View 3 30 Message boxes 11 3 Minimize command 3 30 Minimum 7 36 8 29 Minimum reduction B 9 MinOD 7 28 7 30 7 36 8 29 B 10 MinRFU RLU 8 21 8 22 8 29 B 10 SoftMax Pro User s Manual Mode 4 9 5 10 5 40 Endpoint 4 9 5 10 FLEX 1 4 5 40 Kinetic 4 10 5 11 interval 4 10 5 11 5 44 run time 4 10 5 11 5 44 Spectrum 4 11 5 12 Well Scan 1 4 Modem port 2 16 Molecular Devices Technical Service 11 3 N New command 3 26 9 5 New Window command 3 34 3 36 3 37 3 38 Non Volatile RAM B 10 Normal Display B 10 Notes menu 3 36 Notes section 3 9 3 13 7 47 8 45 text format 9 14 Notes Settings command 3 37 NVRAM B 10 O OD values absolute 7 18 7 31 7 32 8 24 8 25 8 28 8 34 Onset Time 4 10 5 11 7 35 8 28 8 32 Open command 3 26 9 6 Optical density OD 4 39 B 1 B 10 Option button radio button B 10 P Page Setup command 3 6 3 27 7 45 8 42 Parallel Port Windows B 10 Partial plate read 4 18 5 18 5 43 Password 6 7 Password Aging 6 10 Password protection 9 29 author name B 1 changing 9 31 setting 9 31 user name B 15 Paste 9 25 B 4 command 3 28 9 25 Group columns 9 27 pre read data 4 15 PathCheck 4 14 7 26 7 27 Pathlength correction 4 14 7 26 7 27 Peak 8 33 Percent Baseline with Multiplier 8 34 Permissions 6 5 Plate blanks 4 33 4 34 5 30 5 31 B 2 B 3 Plate menu 3 33 Plate section 3 9 3
431. ment Setup dialog box This dialog box allows a user to enter a title for the Statement and to enter text describing its purpose The Statements section is the first section a Document it will appear as the first section in the first experiment hey The user must have permission to create modify electronic state ments to add a statement SoftMax Pro User s Manual 6 17 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator t Electronic Statement Setup y Statement Title Cannes rette emm Apprawal Requesting approval forthe current experiment Cancel Figure 6 10 Electronic Statement Setup Dialog Box This dialog box allows a user to enter a title for the Statement and to enter text describing its purpose After a Statement has been created new statements may be added using the Add Statement button on the Statements section title bar or by selecting Add Statement in the Statements Menu v EA statements E E Approval Requesting approval far the current experiment Statement nat signed Figure 6 11 Statement Section in SoftMax Pro Enterprise Edition SoftMax Pro User s Manual Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator Statements Menu Statements Add Statement Modify Statement Sign Statement Figure 6 12 Statements Menu in SoftMax Pro Enterprise Edition Once a Statement section has been added to the document in the
432. mg oOF Tmax Pro One ar mare points may have been lost during this read Please check your data This error appears only with the SpectraMax Plus Plus 190 340PC Gemini in Kinetic or Spectrum mode VersaMax or FlexSta tion in Kinetic Spectrum or FLEX mode other instruments are unable to continue after generating this error and will simply stop the reading A problem may exist with the instrument or the computer or a communica tion problem may exist between the instrument and the computer This error message means that no signal was recorded for a given wavelength in a given well for an Endpoint or Spectrum scan or at one or more timepoints for a given well and wavelength for a Kinetic run The instrument sent a place holder NAN stands for Not A Number in lieu of a number and con tinued to collect data Depending on the type of assay s conducted it may be very easy or difficult to see which wells were not read During a multiple wavelength Endpoint reading it might be quite easy to spot a missed data point finding one during a long Kinetic reading might be quite difficult however Follow the instruc tions below for the type of reading that was performed when the error occurred in order to assist you in finding lost data points Lost Data with a Multiple Wavelength Endpoint Reading If you get such an error message during a multiple wavelength Endpoint reading try displaying raw signal at
433. microplate chamber of the instrument and buttons allowing you to activate the Read function the incubator Automix and to open or close the drawer s of the instrument note not all functions are available with all instruments Stokes Shift The difference between the wavelengths of the excitation and emission peaks Template Editor A representation of the microplate or a set of cuvettes shown as a grid of wells that can be used to designate the location of blanks standards controls unknowns empty wells or to assign wells to other groups you create The template is a map of the microplate It tells the software what is in the various wells of the microplate or cuvette Threshold Data Display Data is displayed as plus for values above asterisk for values within and minus for values below user defined limits Time at Half Maximum Reduction for a Kinetic or FLEX run that reports the time at half the maxi mum signal within the reduction limits as follows SoftMax Pro first deter mines the point within the reduction limits that has the maximum signal level It then scans the plot from left to right until it finds two points that have signals bracketing one half of that signal value A linear interpolation between these two points is used to estimate the Time at Half Maximum SoftMax Pro User s Manual Appendix B Glossary of Terms Time at Maximum Reduction for a Kinetic or FLEX run that reports the
434. ministrator that user name In previous Windows versions the preferences were saved in the Windows system directory Preinstallation Checklist e Your company s FDA 21 CFR Part 11 team must select a person or team of people to act as the SoftMax Pro Administrator e It will be necessary to know the following information e How many Molecular Devices microplate readers will be connected to computers using SoftMax Pro Enterprise Edition e How many users will analyze data on computers not connected to a Mo lecular Devices microplate reader e How many users will need to review authorize and approve data files e Does your site license s cover the number of computers you will need this many SoftMax Pro Enterprise licenses and people your Enterprise Administrator license must allow for this many users who will be using SoftMax Pro Enterprise Edition Create a list of user names and determine the permissions you will assign to each of them using the sheet provided below This completed sheet may be used as a guide when creating the Enterprise Administrator database and adding users You may wish to add users after setting up templates sets of permissions for groups of users When the same set of permissions will be assigned to multiple users it saves time to create the template of permissions first and then assign a single template of permissions to users Examples of groups of users who might benefit from having templates of
435. mn formulas are long as in this tutorial example they may not be seen in their entirety in the default column size To see the complete formula for a particular column click once on a column to highlight it The formula for that column will be shown in the tool bar Wells Concentration Values MeanValue DE Figure 10 69 Data Group Section with Formula Shown for Highlighted Column 7o Activation Click the button to modify or change a formula Let s look more closely at the formula for 76 Activation to see how it works Double click the Activation column A dialog box called Calculation will appear showing the formula used to derive the data for this column MeanValue Summary 1 100 This formula divides the numbers from the MeanValue column by the value of Summary 1 and then multiplies the result by 100 to achieve a per centage Close this dialog box by clicking either or Cancel and return to the Data section to continue with the Tutorial Resize and then Autosize a Column Position the pointer on the line dividing the columns named Concentration and Values The pointer will change to a double headed arrow Click and drag the line dividing these columns to the right Figure 10 31 shows these actions and the resulting wider Concentra tion column SoftMax Pro User s Manual Chapter 10 Tutorials sioe BEES sac Double
436. mple name will be assigned to the well although the sample name will be included in the group Number of O for Empty and Unknown column formats 1 for other column formats descriptors Descriptor Assigned in the Group Settings dialog box Supported strings are unit ml units mg ml ug ml ng ml mg ng and ml If this column is left blank unit ml is assigned Sample Assigned in the Group Settings dialog box Any text string can be entered descriptor in this field or it may be left blank Assigned in the Series dialog box Can be set to any number or left blank value The tab delimited ASCII files are compatible with spreadsheet programs such as Microsoft Excel which makes it possible export a template from Soft Max Pro import it into a spreadsheet change the template and then import it into SoftMax Pro into the same or a different file Preparing a template file outside of SoftMax Pro for use with the program is not difficult if you use the parameters given above and if you also take into account the manner in which you wish the plate to be arranged For example 9 24 SoftMax Pro User s Manual Chapter 9 File Management and Printing Exporting Graphs Graphs from Graph sections and Well Graphs can be exported in PICT format Macintosh and EMF format Enhanced MetaFile in Windows Graph sec tions can be exported by choosing Export Graph from the Graph menu Well Graphs can be exported by choosing Export Graph fr
437. must assign a fill direction from the top bottom left or right to describe how the series will fill the wells and a number of replicates Various options are diagrammed in Figure 4 29 using a 4 x4 block of wells for more information about replicates see Replicate Samples and Samples in a Series on page 4 30 The maximum number of repli cates depends on the selection of wells if you are filling down from the top for example and you have chosen a block of wells that is 4 wells wide by 8 wells high the maximum number of replicates will be four with the same block of wells selected filling from the left would allow eight replicates If the number of replicates you choose does not divide evenly into the number of rows or columns you select depending on the filling direction the remaining wells that cannot contain replicates will be labeled as additional individual wells in the series For example if you fill a block of 4 x 4 wells from the top and choose three replicates then the first three columns will contain the replicates and the last col umn will contain the additional wells in the series see the last diagram in Figure 4 29 This replicate feature allows you to quickly define the number of replicates and in cases where the series has more samples than a single row or column to name them simultaneously To create a SoftMax Pro User s Manual 4 31 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments
438. n Factor Concentration Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Concentration Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor Dilution Factor 10 10 10 10 10 10 10 10 2 252222523 2225222223 235252252223 2352522525223 2 225222223 2225222223 4 23553555 2 233543245554 2 235534555 mee il 11111111 11111111 31111111 11111111 11111111 41111111 11111111 11111111 11111111 Figure 4 35 Export of the Template Example Shown in the Previous Figure SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Templates can be set up in many different ways If replicates are used as with the example above they can be set up horizontally from left to right as this example shows or right to left or vertically from top to bottom or bottom to top If you are generating template data by means of import from a bar code reader or other source noting the manner in which the data is imported will be helpful in determining the best manner to set up the template Figure 4 35 above shows the first part of the exported data from the template shown in Figure 4 34 In this spreadsheet the first column Group Sample Abbr Units Sample Conc or Name Type Name Descrip
439. n a reduced analyzed form Which reduction options are available depend on the read mode and read type used custom settings using formulas you create are available for all read modes and read types Reduction settings do not affect the raw data but simply cause it to be dis played differently so you may change reduction settings without affecting the raw data in any way Custom reduction formulas require the use of accessors and operators that are understood by SoftMax Pro A list of such accessors and operators can be found in The Formula Reference Guide 6 4 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments The Data Display SoftMax Pro User s Manual After a plate has been read the information sent from the fluorescence instru ment is shown in the data display in the active Plate section The default dis play for data from Endpoint readings raw RFU RLU numeric values Data from Kinetic Spectrum Well Scan or FLEX readings is displayed in plots for mat raw RFU RLU values with respect to time or wavelength and the dis play updates dynamically to show the data collected so far If the instrument is reading more than one wavelength plots for the different wavelengths are overlaid in each well All choices except those defined in the Instrument Settings affect only the manner in which the data is displayed the raw data is not changed permit ting you to o
440. n available a shortcut key combination will be shown to the right of the command in the menu On the Macintosh begin by pressing and holding down the command 3 key followed by the key shown in the shortcut Win dows users should press and hold the control CTRL key followed by the key shown in the shortcut SoftMax Pro User s Manual 3 23 Chapter 3 SoftMax Pro Overview 1 F Some menus are divided into groups of items that perform related functions Dividers are shows as thin horizontal lines between menu items File Items in this menu relate to entire SoftMax Pro files Commands to open print apply password protection and save files are located here Edit This menu contains features for changing items within SoftMax Pro Commands to select copy paste delete and duplicate items are located here This menu also contains the Preferences command which allows you to set options regarding printing communication with the MDC Microplate System instrument and saving files View Choices regarding what is shown and how it is shown are available here The last section in this menu provides a navigational tool allowing you to choose the particular section you would like to view a section chosen in this manner will open if it was closed and will be brought into view within the window Experiment Choices in this menu allow you to create new sections within the current experiment as well as to create a completely new experimen
441. n is shown as a depressed button raw values with a reduced number Click the Reduced button The following options will appear a px Display Raw Reduced Max Min Lr Example Options 46912 HJE s Number d Threshold Ranged Grayscale Figure 10 64 Display Options for Reduced Displays E Choose Grayscale from the drop down menu that appears when you click the Display options menu The dialog box will update to show the low and high limits for grayscale display values which are automatically set to the minimum and maximum data values 10 55 Chapter 10 Tutorials mi Display E xi 2 Faw Feduced hdax Min Lind Example Options High 4 54e4 Low and High ery Low 5 9524 aem Limit Settings Show v ith reduced number Cancel Figure 10 65 Display Dialog Box Updated with Low and High Limit Settings Click OK The dialog box will close and the data display in the Plate section will update to show the data represented by shades of gray as shown in Fig ure 10 66 gt CJ Patent g El Max Min Plate 1 1 2 3 4 5 5 T 8 3 10 11 12 Time 200 secs Interval 2 Secs Reads 101 Ex Em Cutott Lint 485 525 515 Atomi Off Calibrate On Lag Time 0 End Time 200 RE RFU hax 50000 Wavelength Combination Lrn Checksum Failed Plate Last Read 3 11 Phi 127 272004 ded ded ded ded 5ed 5ed S
442. n key Summaries in Group sections can be moved horizontally if desired by drag ging them left or right When you position the pointer over a Group sum mary the cursor changes to show a double headed horizontal arrow Clicking once highlights the summary dragging and then releasing the summary leaves it in the position where the mouse button was released To have summaries line up with particular columns in the Group hold down the control Ctrl key while dragging the summary When you release the con trol Ctrl key the summary will snap back so that it begins flush to the left side of the column under which it was released Each summary can be posi tioned in a different place in the Group section if desired SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments NOH To delete a summary highlight it and press the Delete key or choose Cut from the Edit menu A summary can also be copied or cut and then pasted in a different location You can also change the format of the text in Group section columns and or summaries By selecting the column or summary and then choosing Edit and then Text Style Ctrl T you can change the font size and or style of the text Notes Sections The Notes section allows you to enter text describing the experiment and one or more summary lines that contain formulas To create a summary click the Create Summary button in the tool bar
443. n the Plate section you define a template to be used for analysis determine the instrument settings choose how the data will be displayed and define the type of reduction if any that will be per formed on the raw data received from the instrument 384 Plate 1 T CuvetteSet sections SpectraMax Plus or Plus only determine how the cuvette ee will be read define templates to be used for analysis display data as it is acquired from the instrument define how the data will be displayed and define the type of data reduction The CuvetteSet section can display samples in three different ways three samples per row one sample per row and in a grid of cells which is similar to a microplate format To the right of the data display in the section is a SoftMax Pro User s Manual 3 9 Chapter 3 SoftMax Pro Overview 1 F V Fal StandardCur ve box showing the current instrument settings except incubator settings these are shown at the bottom of the CuvetteSet section after the first cuvette in the set is read Group sections are created automatically when groups are defined in the Tem plate Editor The example shown at left is for a group named Standard Protocol files generally contain default groups you can create others as needed Each Group section contains default columns of data which can also be changed if de sired Within a Group section you can resize columns modify existing columns to use different an
444. name will be different for example group experiment 2 instead of groupQexperiment 1 Any changes made to the template of the destination Plate section will not affect the source section Data from the two Plate sections will not be analyzed together 5 33 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Copying and Pasting Template Regions Contiguous regions of templates can be copied and pasted to other regions of templates within the same or to a different experiment All template settings are pasted into the new template except the group name the new group is given the name Group X where X is the next greater number than that used already for group names To copy a region of a template highlight the region and then select Copy from the Edit menu or press C Macintosh or CTRL C Windows Only the high lighted wells will be copied To paste the region into another area of a template highlight the region and select Paste from the Edit menu or press V Macintosh or CTRL V Win dows Only the highlighted wells will have template information pasted into them Data is pasted using row priority all wells in a row of highlighted wells have information pasted into them before proceeding to the next row from left to right If fewer wells are highlighted for pasting than were copied the data will be pasted until all paste highlighted wells are filled If more wells are highlighted for pasting than were
445. nce within reduction parameters of MinRFU RLU and MaxRFU RLU Minimum Reports the minimum relative fluorescence or relative luminescence within reduction parameters of MinRFU RLU and MaxRFU RLU 6 29 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Lambda at Maximum Reports the wavelength at which maximum relative fluorescence or lumines cence is achieved within reduction parameters of MinRFU RLU and MaxRFU RLU Lambda at Minimum Reports the wavelength of minimum relative fluorescence or luminescence is achieved within reduction parameters of MinRFU RLU and MaxRFU RLU Area Under Curve This reduction estimates the area under the curve as defined by the data plots in wells within the reduction limits The data plots are treated as a series of trapezoids with vertices at successive data points and at the X axis coordi nates of the data points The areas defined by each of the trapezoids are then computed and summed Well Scan Wavelength Combination The default wavelength combination for Well Scan readings is Lm1 and is the only choice besides Custom which can be used to create a different reduction formula Well Scan Reduction Choices are Maximum Minimum Average default and Custom Average provides the average value for all points in the Well Scan 8 30 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Lumines
446. nces dialog box 0 000005 2 17 Reader selection in Preferences dialog box sleleeleeeee eese 2 18 Piters Dialog BOG C ectaet ed dr mio tu ttiig eniti edu di aid ice eueheluun dated 2 19 Autosave portion of the Preferences Dialog Box 00 0 eee eee ee 2 20 Autosave Location Dialog Box Windows sees 2 21 File Prefix Field Windows eeeeee RR IRR Ih 2 22 SoftMax Pro Title Screen Exaniples o mter me mm Rr hee ge 3 3 SoftMax Pro Screen and Window for Macintosh 002 ee eee eee eee 3 4 SoftMax Pro Screen and Window for the PC Windows 0008 3 5 Status Bars for MDC Microplate System Instruments 0 0005 3 7 Tool Bar from an Active Graph Section 0 0 cece eee eee ees 3 11 Example of a Tool Bar Sticking to the Top of the Window 3 11 Closed and Open S6CHONS ssn oaa dc d Ope T oes Ro cR a ora merase ese 3 12 Result of Minimizing an Experiment Tool Bars Hidden 3 12 Separate Dec HO Y WNGOW seses eet eae eee bw epica a machi efus eae 3 13 Example Notes Section Named Results l l 3 13 Plae DeCHON PTT 3 15 Oy OHeDSLDOEIOIN 2550045540445 ETT HOSP ORO IER E deus Pgdq quie 3 17 COU cui C 3 19 uses r 3 21 Menu Bar Macintosh top Windows bottom 0e eee eee eee 3 23 SoMa POMM Bea
447. nd key in conjunction with another key and are shown with the command key symbol 3 followed by the accompanying key To use a shortcut you must press and hold the command key while pressing the second key SoftMax Pro User s Manual HN Keyl Key2 A plus sign between key names means to press and hold down 2 the first key while you press the second key within Windows For 1 7 Chapter 1 Welcome to SoftMax Pro 1 F example press ALT ESC means to press and hold down the ALT key and press the ESC key Then release both keys H Keyl Key2 A comma between key names means to press and release the keys one after the other For example press ALT F means to press and release the ALT key and then press and release the F key The names of keys found on Windows compatible PC computer keyboards vary somewhat from machine to machine The key names shown in this manual reflect typical key names Yours should be sim ilar but may not be exactly like those shown For example the print screen key may be shown on some keyboards as Prt Scrn other keyboards may spell out the complete key name Messages The following types of messages may appear in this manual Ose Calls attention to information that should be followed during instal lation use and or servicing of the instrument or software ANS Indicates a potentially hazardous situation which if not avoided might result in minor or moderate injury loss of
448. nd low limits is assigned proportionally to integer values from 0 through 9 Val ues above the high limit are displayed as a plus and values below the low limit are displayed as a minus Figure 8 7 shows reduced Kinetic data dis played in Ranged format 6 12 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments v Ej Piete 22 m HE per sec oo CDDPBBRPBPBDPBPIPEBPBDBR Kinetic Time 5 00 Interval 023 c pepper Reads 14 Fluorescence Bottom read H a L a Wavelength Combination Lr1 Automix Ott Calibrate on PMT Medium ReadziMel 6 Lag Time 0 00 End Time 5 00 RFO Min RFU Max 20000 max Pls 14 44 Plate Last Read 4 23PM 2 5 2001 B7 O 14 1 2 2 26 5 43 B 48 T a 54 B B Figure 8 7 Kinetic Data Reduced Displayed as Ranged SoftMax Pro User s Manual 8 13 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments k Gray Scale The Gray Scale option presents the raw data in eight shades of gray changing from light for values less than or equal to the low limit to dark for values greater than or equal to the high limit Figure 8 8 shows reduced Endpoint data displayed as Gray Scale v CJ rate AZ Plates Endpoint Fluorescence Bottom read Atomix Ott Calibrate On PMT Auto Wavelength Combination Lr1 Reads Vell 5 Plate Last Read 4 3
449. nd FLIPR files for analysis m sOF Tmax PRO can be found in Chapter 9 of the User s Manual Protocols for drug discovery analysis can be launched from the Assays menu m the Drug Discover folder If You Are Familiar with SOFT max PEG Customize This Default Protocol 1 Delete any sections you do not want 2 Click the Template button and assign any default or new groups as necessary tou can also select replicates there 3 Select the desired Reduction and Display Raw vs Reduced parameters 4 Save thos default protocol with the name Default Protocol in the same folder as the SOF Tmax PRO application you wil be asked if you wish to replace the existing default protocol choose Yes Thos modified protocol will now launch each time you open SOPTmax PRO amp copy of the ongmal default protocol can be found m the Tutorial folder If needed Tutorial 2 ase refer to Chapter 10 in the SOFTImax PRO User Manual D eterrrung the EC 50 using the Wolecular Devices no wash Calion Assay Eitteg FLEX station or FLIPR n associated data file named Tutonal2 pda can be found m the Tutorial folder of FT max PROS MDC Product Humber R 5035 Product Mame FLIPE Calion Assay Kit Initial alume LOO UI of Cells spiked with 100p of Agorust Concentrations that need to be assigned in the Template increasing concentration from left to right Columnl uM calumnz 0 01uM Columns 05uM calumn4 O 05uM Columns d 5uM Columns 1 uM Calumn
450. nding on the setting cho sen for either a 96 or 384 well plate type Setting this value to a positive number assists in ensuring that aspirated fluid does not drip while the tips are being moved during an injection helping to prevent contamination With subsequent transfers you will need to calculate the volume of fluid within a well to determine the value to assign for the air gap setting to avoid tip contact with fluid in the well Recover FlexStation only This menu item can be used to reboot the FlexS tation in the event of a fatal error encountered typically while fluid re mains in the tips More information regarding the function and appropriate times to choose this menu item can be found in the Flex Station instrument manual Assays Menu Set Folder protocol 1 Set Folder Allows you to select a folder or subdirectory containing com monly used protocol files In addition to any protocol files residing within this folder which will be listed below the dividing line and may be opened by clicking them an additional level of subfolders if any will be shown below a center division Protocol files can be used repeatedly for specific types of assays for example These files can be named in any way you choose Folder name s after dividing line Choosing any of these folders will open a submenu showing protocol files residing in this subfolder Select one of the protocol files to open it File name s af
451. nding upon which experiment is active For example if the experiment labeled Experiment 1 is active the Edit menu will show Delete Experiment 1 Using the Macintosh you can also delete the selected experiment by pressing 3 within Windows you can press CTRL Take care before deleting experiments that they do not contain infor mation you wish to save You may delete multiple experiments simultaneously by holding down the Shift key and activating the tool bars of the experiments you wish to delete Having done this the Edit menu will now show Delete Selection instead of Delete Experiment You can add new sections to the active experiment using the New Section command under the Edit menu Sections There are five types of sections Notes Plate Cuvette Group and Graph A brief synopsis of these sections is given below Notes sections are used to record information pertaining to the experiment You NE Intraduction P T can enter text in this section as you desire in addition you can create summaries containing formulas for displaying reduced data Text in Notes sections can be for matted font size and style any way you choose Each Plate section shows a grid of cells which corresponds to the wells of the mi croplate To the right of or underneath this grid is a box showing the current in strument settings except incubator settings which are always shown at the bottom of the Plate section after data is acquired I
452. ned it is automatically subtracted To see the reduced number without the group blank subtracted mask the group blank wells Group blanks apply to all wells in a group and thus may be subtracted from wells in more than one Plate section Blanks in Combination SoftMax Pro User s Manual It is possible to use blanking functions in combination You could assign a blank group in the template and also include blank wells within different groups The effects of the different blanking functions are cumulative so it is important to understand the function of each before using more than one type of blanking in a single assay Plate blanking is applied to the raw data 5 31 Chapter 5 Reading Using Fluorescence or Luminescence Instruments 5 32 Blank Group Plate Blank Group Associated Blank Wells N acquired from the instrument whereas group blanks are applied to the reduced number Group blank subtraction is only visible in the Plate section when the reduced number is displayed Figure 5 31 shows the two types of blanks in the Template Editor Experimentz1 Plate 1 Group Sample Tror Mr Ber ies wf Assign Clear Figure 5 31 Template Editor Showing Plate Blank and Group Associated Blank Wells In the figure above note that the plate blank in the upper left appears as a separate group named Blank and individual samples within this group are named BL The group associated blank wells shown in th
453. ng e Long Private Declare Function RegisterWindowMessage Lib user32 Alias _ RegisterWindowMessageA ByVal mgeName As String As Long Const WM SETTEXT 12 define a windows message value Function SendMsgToFro ByVal msgStr As String As Long hWnd FindWindow SOFTMaxProMainWnd SoftMax Fro If hWnd O Then one beep means Fro ie not running Beep Else Fro ie running eo use the SendMessage AFI softmaxMsg KegieterWindowMeeeage SOFTMaxFroMeqg If softmaxMsg O Then 12 14 SoftMax Pro User s Manual Chapter 12 SoftMax Pro Remote Command Language This version of Pro does not understand external msgs Deep Deep Elee SendMessage hWnd WM SETTEXT softmaxMsg msgStr End If End If End Function This ie an example of how to use the above routine Sub SendNewMegToFro SendMsgToFro New End Sub MFC C Interface to SoftMax Pro Remote Commands that Return Values SoftMax Pro User s Manual Some SoftMax Pro IPC commands return values By default they put the data in the clipboard but optionally they will use the WM_COPYDATA mecha nism to return data directly to the caller In order to have the SoftMax Pro response returned in this fashion the com mands must be sent using WM_COPYDATA so that SoftMax Pro knows what to send the response to SoftMax Pro will then reply with another WM_COPYDATA message that contains the response MFC Example Code Global variable used in holding ID de
454. ng performed is selected the alert shown on the right side of Figure 4 40 appears A similar alert is shown if you choose either read function with more than one cuvette selected asking if you wish to replace the data or reference for all cuvettes Replace data in Al Heplace reference in selected cuvettes Cancel Replace Cancel Replace Figure 4 40 Alert Regarding Replacing Data for CuvetteSet Section Note the alert box presented depends on the number of cuvettes in the CuvetteSet sec tion and whether or not one or more are selected at the time you choose to read again For Kinetic and Spectrum modes the raw data plot is automatically displayed as a graph in the cuvette You can also enlarge the view of selected samples by double clicking by using the Well Graph button on the CuvetteSet section tool bar or by choosing Graph from the CuvetteSet menu The instrument settings table shown on the right of a CuvetteSet section is similar to the one in a Plate section Automix is not available for cuvettes Instrument settings must be the same for all cuvettes in a CuvetteSet section CuvetteSet sections have a limit of 96 cuvettes Should this limit be reached the New Sample sub menu of the CuvetteSet menu and the corresponding tool bar button will become unavailable dimmed and it will be necessary to create a new CuvetteSet section General Information about a Reading SoftMax Pro User s Manual After beginning a rea
455. ng that are under this limit will not be shown and will be excluded from data reduction The default is 0 RFU RLU Max RFU MaxRLU The limit for the maximum value you wish to report Any values from the reading that are above this limit will not be shown and will be excluded from data reduction The default is 20 000 RFU RLU Start Wavelength nm Specifies the limit for the minimum wavelength setting you wish to report Any values from the reading that are under this limit will not be shown and will be excluded from data reduction End Wavelength nm Specifies the limit for the maximum wavelength setting you wish to report Any values from the reading that are above this limit will not be shown and will be excluded from data reduction NOINA The settings for the Start and End wavelength settings cannot be below the lowest wavelength or above the highest wavelength entered in the Instrument Settings for the Spectrum reading Spectrum Reduction SoftMax Pro User s Manual The Spectrum reduction formula is applied to the list of numbers in each well values at each wavelength after the wavelength combination formula is applied The default reduction for Spectrum readings is Lambda at Maxi mum You can choose Lambda at Minimum Maximum Minimum or Area Under Curve instead from the pop up menu or you can choose Custom and create a different reduction formula Maximum Reports the maximum relative fluorescence or relative luminesce
456. ngle well it would be necessary to take a ratio Lm1 Lm2 of values that have been read at two different points in time To solve this problem you may select the Interpoate Raw Data checkbox in the Reduc tion dialog box Note that this checkbox is shown only when two or more wavelengths are present in the reading Enabling this checkbox causes SoftMax Pro to use quadratic interpolation to calculate the interpolated values This results in the loss of one point at each end of the plot Prior to beginning the calculation SoftMax Pro determines the starting point by averaging the initial data points of the separate wave lengths For example if the first data points of the two wavelengths have time values of 0 3 and 0 5 seconds SoftMax Pro will average these and use 0 4 sec onds as its initial point for interpolation Time secs o A2 Lm1 O A2 Lm2 25 Time secs Oo A2 Lm1 Oo A2 Lm2 Figure 8 22 Top Raw Data without Interpolation Bottom Interpolated Data Custom Reduction Formulas SoftMax Pro User s Manual If the predefined reduction formulas do not meet your needs you can create one or more different reduction formulas for any or all read modes Endpoint Kinetic Spectrum Well Scan or FLEX Choosing Custom from any of the menus or dialog boxes causes a Formula button to appear an example of this 8 35 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments button in
457. nother or from one experiment to another rather than taking time to recreate it Summaries can also be copied and pasted within SoftMax Pro After pasting both formulas and summaries can be edited Contiguous column formulas may be copied and pasted together Summaries must be copied and pasted individually To copy a formula or summary highlight it and then select Copy 3 C CTRL C from the Edit menu Activate the area or section in which you would like to paste it if necessary with a formula access the particular dialog box into which it will be pasted After placing the cursor in the correct location and highlighting any existing information to overwrite it select Paste 38V CTRL V from the Edit menu The pasted formula or summary will appear in the new location Formulas and summaries can be deleted by highlighting them and pressing the Delete key or choosing Cut from the Edit menu Duplicate and New Duplicate SoftMax Pro User s Manual All sections and even complete experiments can be duplicated by making the section or experiment active and then selecting Duplicate from the Edit menu All of the information in the section or experiment including data is copied to the newly created section or experiment The name of the new section or experi ment is the same as that of the original with the addition of the word Copy e g OldName Copy Duplicated sections are created at the end of the exper iment Duplicated e
458. nsfer 2 Transfer 3 pU Transfer Settings qnenesesseanenesennennsns tnsinsesumaseneanaseseuansnnensenanasssenenssnetemncsnanansesessissenesanacons Pipette Height l P 3 oo Time Point secs Valume 1 Minimum time 14 secs Figure 5 45 Compound Transfer Settings Box Assay Plate Fluid The entry in this box should be equal to the largest initial volume for any well in the plate SoftMax Pro assumes all wells hold the same initial volume This setting is used to compute the total volume that will result in each well after all fluids have been dispensed This calculation is made in order to warn you regarding potential overflow of fluid from the wells The intial volume entry has an integer range of 0 to 999 in microliters You are not required to enter anything in this field Any value entered is saved with the file but the value is not displayed anywhere except in the Compound Transfer Tab setting Transfers You may enable up to three compound transfers during a reading or choose none When transfers are enabled color coded transfer buttons appear on the Instrument Settings dialog box Selecting one of these buttons allows you to set the parameters for that transfer Settings for each transfer include Pipette Height from 1 to 999 uL This setting determines the amount of fluid in microliters measured from the bottom of the microplate well above which the tip of the pipette will remain This setting helps to ensur
459. nstrument if it is not already chosen and then close the dialog box A synopsis of the steps you will take during this tutorial is given below Create a new file Instrument setup Template setup Set reduction parameters Set display parameters Save the protocol Read the plate you won t actually do this instead open a saved file Analyze data graph of concentration vs RFU Print the report CoU ops T Hs s When you install SoftMax Pro you are asked to choose between absorbance fluorescence or FlexStation installation The choice made at that time will deter mine the type of default protocol file that is installed Tutorial 1 found at the start of this chapter pertains to users of either absorbance or fluorescence instruments this tutorial Tutorial 2 deals with the FlexStation instrument only and FLEX mode in particular 10 35 Chapter 10 Tutorials Description of the Assay Example Samples in units ml Curve Fit Instrument Settings Read Mode Wavelengths Auto Cutoff Sensitivity liming Automix AutoCalibrate Assay Plate Type Wells to Read Compound Source Compound Transfer Triturate Pipette Tips Layout Following are the details about the EC50 assay settings we will use to create a protocol file using SoftMax Pro 0 0 01 0 05 0 5 1 5 10 and 100 each standard run in duplicate 4 Parameter FLEX One Excitation 485 nm Emission 525 nm Enabled
460. nstrument such as a robot may be competing for the use of the serial port to which the MDC instrument is connected Try uninstalling the software for any of these items and or ensure that they use a different serial port from that used by the Molecular Devices instrument b If no instrument is connected to the computer be sure the serial port setting Edit gt Preferences in SoftMax Pro is set to None 4 If your files contain many Plate sections or if you are using a FlexSta tion protocol try suspending recalculation Edit gt Suspend Recalcu lation to free up resources on your computer 11 15 Chapter 11 Troubleshooting 1 3 General Problem Insufficient Memory in a Macintosh 1 If errors occur while using SoftMax Pro for the Macintosh it may be that the amount of memory allocated for the program is insufficient Two items need to be checked the amount of memory your Macintosh has available for use and the amount of memory allocated to SoftMax Pro To see the amount of memory available within the Macintosh first quit all open applications then go to the Finder and choose About This Macintosh from the Apple menu File Edit View Special About This Computer The information box that opens will list the amount of memory used by the System file and the amount of memory remaining under the heading Largest unused block that can be assigned to applications such as SoftMax Pro The amount
461. nstruments Well Graph Options SoftMax Pro User s Manual Along the bottom of each Well Graph dialog box are eight buttons five when using Well Scan mode that perform the following functions e Print Opens the Print dialog box e Reduction Opens the Reduction dialog box By default the ranges on the enlarged plot are set to the limits defined in the Reduction dialog box and the information included near the bottom of the window is based on the reduction settings currently in use You may change these settings wavelength combination and type of reduction used minimum and maximum value limits lag time and end time and number of Vmax Points More information about the reduction options can be found later in this chapter Changes to the reduction settings made within well graphs will be applied to all wells in the Plate section When viewing Raw data you can scale the graph to see data outside the reduction limits line s are included on the graph indicating the limits and the area outside of them will be shaded slightly This view should assist you in deter mining the best limits for the reduced data Show Raw Show Reduced button This button toggles the Well Graph between views of raw and reduced data When the Well Graph is opened the initial view depends on the Dis play settings for the Plate section If raw data is being displayed in the Plate the initial view in the Well Graph will be raw data and the butt
462. nstruments SoftMax Pro allows you to create files containing all of the parameters required for acquiring and analyzing data from Molecular Devices Microplate System instruments You may also import data from other Molecular Devices instruments such as Analyst and FLIPR systems for dis play and analysis SoftMax Pro is available for Power Macintosh computers using OS 8 6 or greater OS X in Classic mode only and PC compatible computers using Windows 2000 NT 4 x and XP SoftMax Pro is powerful yet easy to use It performs three major functions e Instrument Control SoftMax Pro allows you to set up and run a complete assay Instrument settings can be saved as a protocol file and used repeat edly for reading different microplates All stand alone instrument functions can be controlled using the software In addition SoftMax Pro provides capabilities that are not available when using MDC Microplate System instruments in stand alone mode for example user defined Kinetic run times read intervals Automix parameters and more Data Collection and Display SoftMax Pro collects and stores all raw data received from the instrument Data is displayed on the computer screen in a grid format that corresponds to the wells in a microplate all instruments or individual cuvettes SpectraMax Plus or Plus only How the data is shown depends on other settings made using the software In addition to displaying data collected from an MDC Microp
463. nstruments To learn more about using FLEX mode with the FlexStation instrument go to Tutorial 2 on page 10 35 Hard and Soft Parameters The only settings that must be defined before reading a microplate are the instrument settings found in the Instrument Setup dialog box These set tings tell the instrument what conditions should be in place in order to collect data They include the read mode Endpoint Kinetic Spectrum or Well Scan the number of wavelengths specific wavelength settings and so on These settings called hard parameters define the conditions under which the data is obtained Once a microplate has been read the instrument settings cannot be changed without deleting the data Soft parameters include the template definition and data reduction settings These parameters can be defined prior to reading a microplate but they are applied only after the plate is read and can be changed at any time In this tutorial you will build a complete protocol based on the settings found in the Default Protocol file which will include both hard and soft parameters You will save the completed protocol file prior to collecting data this file could then be used to generate future data files You should ensure that the instrument that is connected to the com puter is chosen in the Preferences before proceeding in the Edit menu choose Preferences In the dialog box that appears choose your instrument if it is not al
464. nt is connected to the computer or if no instrument is connected by which instrument has been chosen in the Reader section of the Preferences dialog box and the particular settings chosen for that instrument The minimum interval for SpectraMax and VersaMax instruments depends on the number of wavelengths the Auto mix status the number of strips and the distance the monochromator and fil ter wheel of the instrument must move The minimum interval for Vmax UVmax and ThermoMax instruments depends on the number of wave lengths and the Automix status If you set the interval time to be shorter than the instrument s minimum read interval the program will alert you with a message Additionally with SpectraMax and VersaMax instruments an inter val that is too short will cause a warning message to appear telling you that no calibration can occur between reads If you are writing a protocol that will be used with a different instru ment other than the one connected to the computer or chosen in the Reader preferences you can disregard the alert and save the setup However if you attempt a reading using a protocol which has set tings that are not correct for the particular instrument you are using an error message will instruct you to change any setting that is out of range before a reading can be initiated SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments NOFA To achieve the shortest
465. ntains the Series and Assign buttons SoftMax Pro User s Manual Figure 5 26 Assign Area of the Template Editor Tool Bar Assign Button and Function Click this button to apply a sample name to one or more wells that have been selected in the template You may also perform this operation by using the Enter key on your keyboard Series Button and Function This button is used to define several samples as a series allowing you to easily enter incremental sample descriptors for example dilutions or concentra tions and sample names to the template as long as the increment can be expressed as a mathematical operation Replicate Samples and Samples in a Series You can assign the same group or sample definition same sample name and description within a group to multiple wells to create replicates For example you might want to read standards in replicate to ensure that anomalies can be 5 27 Chapter 5 Reading Using Fluorescence or Luminescence Instruments excluded prior to generating a standard curve Replicates can be created using the Assign button or with the series function Creating a series is faster than assigning replicates individually The series option can be used as long as the replicate values conform to a pattern that can be described by a mathematical operator multiply by 2 or add 5 for example A series is defined in one direction starting from left right top or bottom and therefor
466. ntered this name will be available for use in that experiment thereafter New groups can be created independently from selecting or assigning any wells cuvettes in the Template Editor Each well cuvette designated as being part of a group has associated with it a group name a sample name or replicate ID a sample descriptor optional and a default column format group type such as Standards for example When a group is applied to selected wells or cuvettes the group name is dis played in bold type over the wells cuvettes and the wells cuvettes bear the same color SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Template Editor Tool Bar Group Drop Down List Microplate or CuvetteSet Map Sample Descriptor Field SoftMax Pro User s Manual Clear Edit Sample Series Assign Button Button Drop Down List Button Button 3 Sample pet Im Baken Clear uu Group Standards w pou pus Figure 4 17 Template Editor Dialog Box Groups are always visible both in the Template and in the data display of the Plate section or CuvetteSet section Even though they are grouped single wells or cuvettes are still selectable in either the Template Editor or the data display Samples and groups may exist across multiple Plate sections or CuvetteSet sections as well as within a single Plate section or Cuvett
467. ntly running this will return a null value thus requir ing your program to discontinue until SoftMax Pro is restarted HWND hwnd FindWindow SOFTMaxProMainWnd SoftMax Pro Get your window handle This is the window to receive the return mes sage HWND MyWnd GetSafeHwnd Assign which command you wish to send Place the command in heap space which appears to be necessary with older Windows operating sys tems char cmdStr Read Example remote command to send char msgStr _strdup cmdStr String duplication copy command to heap space e Create the Win32 COPYDATASTRUCT message that will be sent See Microsoft documentation regarding this structure for details SoftMax Pro User s Manual 12 5 Chapter 12 SoftMax Pro Remote Command Language 12 6 COPYDATASTRUCT cd This struct is defined in Microsoft include file cd dwData DWORD MyWnd Handle to a window receiving return information cd cbData strlen msgStr 1 Length of message string adding 1 for null terminator cd lpData msgStr String pointer to remote command Finally send the message to SoftMax Pro A complete of list of remote com mands can be found in SoftMax Pro Commands on page 12 9 Don t for get that the tag value from RegisterWindowMessage above is sent with the command string See Microsoft documentation regarding the SendMes sage function for more information SendMes
468. nu 5 33 Select echon Dialog DON oes ud equ dem ued d a dV RHREE UP RUN quie merde iud 5 35 Alert Regarding Replacing Data for Plate Section 0 000 006 5 35 Status Bar during a Kinetic Reading SpectraMax 0 0000s 5 35 Instrument Settings Dialog Boxes for FLEX Mode 0 0 0 0 0005 5 39 Instrument Settings Dialog BOX 52 2irini prb qaot ert Rp Se RR Fe ac ara ce is 5 40 Wavelens HS IBS cose ma menge adesset Eg ds Idus dui duis 5 42 Readies Sele aera eer ANE quded quat edebat odd d dedit sean 5 42 DNLESSHSIBVID OCE cedro nsec EER dom q y dp 9 Oe aparato 5 43 Wells to Read Section of the Instrument Settings with Wells Selected 5 44 PLEX Timing 5ettinps BOX vss uasuotnp tre birth E Spears mu aac R ani ga brain odes 5 44 PULOMIX SOUS DON veo ue quc 9 aac dcs dinde ed pde quond ed ha d vale sq uq e 5 45 Compound Source Sein gS BOX iue aeria doo aksa RR Pp n c eee reed 5 45 Compound Transfer Settings BOX eee 5 46 Triturate oeltnps BOX stesen ves ered op See ex oy deren mda wa PN dpa Putas 5 47 Pipette Tips Layout Settings BOX s ea x xe e x mdp den d Papin incra Rea 5 48 Compound amp Tip Columns Settings Box 1 0 0 eee cece eee 5 49 Select cechom Dialog DON oque d ade e Had aH ur qr Cm Ug UP o ae E E R 5 51 Alert Regarding Replacing Data for Plate Section 00006 5 52 Status Bar during a FLEX Reading vaso ss s2d erp yop a TREAT TOV a edm ET A ars 5 52 Crea
469. nu item does not appear Template Opens the Template Editor The Template Editor allows you to describe the samples that are in the wells thus providing the link between the raw data and the analysis groups You can create new groups in the template or assign cuvettes to already established groups If your experiment contains multiple CuvetteSet sections the tem plate for each CuvetteSet section must be defined using the Template Editor in that section You can create a new template or modify an ex isting one using the Template Editor After groups are defined in the template and you return to the Cuvet teSet section the data display will show the cuvettes that are assigned to groups by means of colors in their locator headings if viewing by one or three cuvettes per row or as colored outlines if viewing in a grid if you have a color monitor or in shades of gray on a mono chrome monitor Button Equivalent Template in the CuvetteSet section tool bar Reduction Opens the Reduction dialog box Options available in the Re duction dialog box depend on the type of reading performed End point Kinetic Spectrum Well Scan or FLEX and the instrument used to collect the data The Reduction dialog box is used for initial reduc tion of the data collected from the microplate reader The optical den sity information collected is reduced to a single number per well Button Equivalent Reduetion in the CuvetteSet secti
470. number of readings e Read type fluorescence and also bottom read e Assay plate type manufacturer and number of wells e Automix e Wells to read e Compound source e Compound transfer e Triturate e Pipette tips layout e Compound and tips columns e AutoRead No information is provided here regarding operation of the FlexSta tion instrument If you need information about the function of the instrument itself and how to set it up for a reading consult the man ual that accompanied the instrument Information here concerns only the SoftMax Pro interface and how to use the software to perform a reading Settings for the reading are made in the Instrument Settings dialog box which can be accessed in three ways 1 Choose Instrument Setup from the Control menu 2 Click the Setup button in the tool bar of the active expanded Plate section or 3 Double click anywhere in the settings box located to the right of the well grid in the active expanded Plate section You may create more than one Plate section in a single experiment and the instrument settings associated with each Plate section can be different Instrument Setup can only be chosen while a Plate section is active and the settings you choose will affect only that section A new Plate section that is created in an experiment that contains an active Plate section will contain the same instrument settings found in the previously active section The settings for the n
471. o PPM WC V ad e epu dne 8 44 Graph Options Dialog Box ssseeeeeeeeeeee e 8 47 Standards and Unknowns Groups Created 1 0 0 0 cee eese 8 48 otandard2 Group Ge HO saute at add guise dapeededszeu pice a anaes 8 48 Graph Options Dialog Box with Plot Name Changed to Standard 1 8 49 Graph Options Dialog Box with Second Plot Added for Standard 2 8 49 Graph Displaying Three Standards Plots 0 cee eee eee eee ee 8 50 Plot Showing Error DAS Co sacas eni RATE eee oe eeeotle Seq eod EE NEUEN 8 5 fcis nudis P m 8 51 Graph with nci T 8 53 Cra phi itt 5er Log Wit een dae nues e wies esas pause mda s da pia pint estes 8 54 Graph with Log Log Fit 22262 eottpOpRRU rediet inida bd eed ded ird 8 55 Graph with Quadratic Fit ac a odor E eeboriete drpacs exon peur p dew dos dnbie see 8 56 Graph with 4 Parameter Fit lees 8 57 Graph Witt Eos Loglt BIE ius sa casu seme pd cases eds 2E d add ne id duiracdos 8 58 Graph with Point to Point Fit 0 0 eee eee eee re 8 59 Graph Witt Cubic Spline Fit cscnncsteeveceeeeanreeeneates ase ean pd pies ads 8 59 Graph with Exponential Fit eee ce eee ree 8 60 Graph Options Dialog BOX sce o axes wb sibus aa mde da PR MEE Padus 8 62 Scatter Graph with Points Connected Linear Fit Applied 8 63 Bar Graphs Cluster and Stack 8 63 26085 1018 IOS DOX caedere m eune xev aucun abere qid did apt esas ANS adam s modis 8 64
472. o User s Manual In wavelength priority the instrument will read all wavelengths for the first column of wells in the microplate and will then read all wavelengths for the second column etc The table below shows the priority settings by MDC Microplate System instrument for the different read modes Table 4 3 Column vs Wavelength Priority SpectraMax Plus User choice Wavelength Column VersaMax Plus SpectraMax Plus 4 SpectraMax 190 190PC Column Wavelength Column SpectraMax 340PC 340PC 4 SpectraMax 250 Column Column Column SpectraMax 340 VersaMax ThermoMax Column Column UVmax Vmax Emax Column 1 You can choose between wavelength and column default priority readings in the Instrument Settings dialog box Data Display During a Reading The values read by the MDC Microplate System instrument will appear in the data display of the Plate section or CuvetteSet section as they are received from the instrument in real time Depending upon the read mode Endpoint Kinetic or Spectrum the number of wavelengths the plate type and the dis play options selected the data display will show one or more numbers or plots You can enlarge the display of one or more wells while the a Kinetic or Spec trum reading is in progress Double click a single well to enlarge its display to a Well Graph or hold the Shift key while selecting more than one well and then choose Graph from the Plate menu or click the graph
473. o determines the parameter val ues that best fit the data The function with these parameters is then drawn on the graph Ideally the type of fit used should be determined by the underly ing chemistry of the assay and could be set before data is read A good discussion of curve fitting appears in Data Analysis and Quality Control of Assays A Practical Primer by R P Channing Rogers in Practical Immuno Assay edited by Wilfrid R Butt published by Marcel Dekker Inc New York 1984 When a fit is chosen the Graph section is updated to show the coefficients describing the fit when applicable These values are displayed in the legend at the bottom of the graph Figures 7 43 through 7 50 show the same graph with the different fit algorithms applied Note that the legend information changes along with the formula for the fit at the bottom of each Graph section for each different fit All plots on a graph must have the same type of fit When a curve fit is chosen the values for the coefficients of the equation A B C etc will be shown as will the square of the correlation coefficient R 2 The correlation coefficient describes how well a change in x values correlates with a change in the y values Please see Judging a Good Curve Fit on page 7 64 for more information The R 2 value should only be used for curve fits having linear axes SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instrumen
474. o the Notes section at the appropriate place than one page The bottom line of the first page and the top line of the second page are partially printed When printing a well graph If you enlarge the well graph to greater than 12 inches the graph will part of it is missing not fit on an 8 5 by 11 inch page and the printout will be truncated Reduce the size of the graph and print again 11 14 SoftMax Pro User s Manual General Problems Symptom Data from a CuvetteSet section reference or reading doesn t seem right Macintosh bombs or gives errors that cause Soft Max Pro to crash 3f Double clicking a file with the pda extension launches the Printshop application T Files disappear from the list of saved data or proto col files or cannot be opened file name extensions are not saved properly SoftMax Pro runs very slowly or or stalls completely SoftMax Pro User s Manual Chapter 11 Troubleshooting Possible Cause Solution Check that the cuvette is oriented properly in the cuvette port of the instrument the clear sides of the cuvette must face left and right when you are in front of the instrument If the cuvette is oriented improperly remove it replace it in the correct orientation and repeat the reference or reading The amount of memory available in your Macintosh or the amount of memory allocated for SoftMax Pro may be insufficient See General Problem 1 below for more inf
475. of memory available should be equal to or greater than minimum amount required by SoftMax Pro Yerasion Mac 03 9 0 Built in Memory 192 MB Virtual Memory 193 MB used on Hard Drive Largest Unused Block 638 5 MB amp i Apple Computer Inc 1983 1999 EJ Mae 03 35 MB Figure 11 2 About This Computer Dialog Box If you open many files simultaneously or if you open SoftMax Pro while other applications are open in the background memory errors can occur Also if you make a Kinetic run with a large number of data points you may not have enough memory with a typical allocation SoftMax Pro requires a minimum of 7223K over 7 megabytes of memory to run but this is not opti mal The preferred size is 8223K over 8 megabytes or larger Try increasing the amount of memory allocated to the program To do this quit SoftMax Pro if it is running Then highlight the SoftMax Pro program icon click it once and then choose Get Info 1 from the File menu The dialog box that opens lists suggested and minimum memory allocations see Figure 11 3 11 16 SoftMax Pro User s Manual t Chapter 11 Troubleshooting EAM FF Inf SS Uy SOFT Mae PRI 61993 2001 Molecular Devices Corp Kind application program Memory Requirements Suggested Size 10000 K Minimum Size 8000 K Preferred Size 10000 K Hate Memory requirements will increase by 2 152 E if virtual memory is turned off _ Lo
476. of the SoftMax Pro window including experiments and sections e the menus and tool bars found within the SoftMax Pro program e the basic steps required to perform a reading complete information regard ing reading using SoftMax Pro can be found in Chapters 4 or 5 choose the chapter that is appropriate for the instrument you are using and e the basic types of data analysis detailed information regarding data analy sis can be found in Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using SoftMax Pro employs standard Macintosh and Windows conventions for using menus dialog boxes windows and the mouse This manual assumes that you are familiar with the basic operation of your computer While some general instructions are given here and many of the terms used in SoftMax Pro are defined in Appendix B Glossary of Terms if you need additional assistance regarding the basic use of your computer please review the docu mentation accompanying it The SoftMax Pro Window SoftMax Pro User s Manual When you start SoftMax Pro a title screen will appear showing the informa tion entered during registration along with the SoftMax Pro version the instrument type if connected the ROM version installed in the instrument if available and copyright information P SOFTmax PRO Drug Discovery Edition Pi SOFTmax PRO Life Sciences Edition Figure 3 1 SoftMax Pro Title Screen E
477. of the SoftMax Pro window is a status bar that shows the type of instrument that is connected to the computer or selected as the Reader type via the Preferences menu see Figure 3 4 Current Chamber Click Instrument Temperature to start Ref Incubator Automix Drawer Icon Display areading Button Button Button Button w Y Y SpectraMax Plus Plus 9 CuvetteSet Section Status Par FlexStation T EE NENNEN Status Bar D lm Bel VersaMax and SpectraMax Plate Section Status Bar ThermoMax Status Bar eeta lods E Emax Vmax and UVmax Status Bar Figure 3 4 Status Bars for MDC Microplate System Instruments The status bar shows some or all of the following items depending on the type of instrument connected or chosen from left to right Instrument icon An icon such as that shown at the left indicates that the instru ment is communicating properly with the computer If an X appears on top of the icon the computer is not making proper contact with the instrument NO Double clicking the instrument icon opens the Preferences dialog box 21 2 C Temperature display With ThermoMax VersaMax SpectraMax or FlexStation instruments a small window after the instrument icon shows the current temper ature within the microplate chamber in degrees Celsius With the SpectraMax Plus and Plus 9 the SoftMax Pro status bar shows the temperature within th
478. oints option connects the points in order of appear ance by sample name not the appearance of points from left to right on the graph 6 62 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual Graph Options Button v X and Y Axis Buttons B concentration y A Bx A Plot Groupz1 concentration vs Mean y alue Figure 8 50 Scatter Graph with Points Connected Linear Fit Applied Other options in this dialog box allow you to plot the data as a bar graph with data shown in clusters or stacked Figure 8 51 shows both of these bar graph options Fit Nort v Graph 1 7 Graphit Graph 2 0 4 5 Sample Number E Plot 2 Group 2 Samples vs Mean value E Plot 1 Group 1 Samples vs Mean alue eee mmm A A A Plot 3 Group 3 Sample vs Mean alue Cluster Bar Graph p MRAR A A CA E Stack Bar Graph Sample Number E Plot 1 Group 1 Samples vs Mean alue Plot 2 Group 2 Sample vs Mean value A Plot 3 Group 3 Samples vs Mean alue Figure 8 51 Bar Graphs Cluster and Stack NOH Dar graph options for multiple plot graphs are only available when each plot contains the same number of data points and has matching X axis coordinates 93831 When choo
479. om Molecular Devices The list box displays user privileges that are avail able with the selected software This list refreshes each time the software pro gram selection is changed Checking the checkboxes before items in the list box allows the administrator to assign specific privileges to the new user The privileges can be changed later from the Modify User dialog box SoftMax Pro User s Manual 1 k Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator SoftMax Pro User s Manual After assigning user privileges click the Finish button The new user will be displayed immediately in the registered user list box on the Users property page Note that the Modify User and Remove User buttons are both disabled These buttons are enabled only if a user is selected highlighted in the Users list box on the Users property page 6 11 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator 6 12 Privileges When adding a new user to an Enterprise Administrator database after defin ing a user name and password the administrator will be prompted to define privileges for each user User Privileges BS xi The Following privileges are avalable For the selected software type Assign permissions by either choosing predefined template or selecting manually Choose Template x SoftMax Pro C Save files and protocols Sign e statements a Create
480. om the Plots menu that appears when a Well Graph is opened Copying and Pasting Parts of an experiment can be copied within a SoftMax Pro file to or from another SoftMax Pro file or to or from another program using the Copy and Paste commands Plate section and CuvetteSet section data and text and summaries in Notes sections can be copied and pasted to other sections within the same or a different experiment within SoftMax Pro or to other pro grams Virtually any ASCII formatted text can be copied and pasted into a Notes section A graph can be copied and the resulting picture pasted into another application Template information can be copied and pasted from one part of a template to another within the same or to different templates and from one experiment to another Plate Section and CuvetteSet Section Data and Templates Copying and Pasting within SoftMax Pro SoftMax Pro User s Manual Data can be copied from one Plate section to another or from one CuvetteSet section to another within SoftMax Pro Make the source section active by clicking on it and then choose Copy 3 C CTRL C from the Edit menu Create a new section if necessary and make the destination section active Choose Paste 38V CTRL V from the Edit menu to paste the data into this section When copying Plate sections all well data is copied It is not possi ble to copy and paste the data from only certain wells Even if you select only one well prior to choos
481. omplete agreement between the User and MDC with respect to the Software and the License which agreement supersedes any proposal or prior agreement whether oral or written and any other communication between the User and MDC relating to this License Agreement lU SOFTmax PRO User s Manual Contents T SoftMax Pro User s Manual Ij rr ee ee ee ee ee ere ee 1 xi jr PPM 1 xix Chapter 1 Welcome to SoftMax Pro 2 eee ee eee eee 1 1 TPH OCUCHON PEL 1 3 New to SoftMax Pro 2 40 44 sed doi fcd Reed dixi dah ae eke ee 1 4 What s New in SoftMax Pro Version 4 3 0 0c eee eee eee eee 1 4 About the Documentation 0 00 eee e eee eee eres 1 6 Conventions Used in This Manual 002005 1 7 Sete Help serii 4cudt beset eee heen shoes aeetet eae see 1 9 Macintosh On Line Help 0 cee eee eee eee eee ee 1 9 Windows On Line LIelD uuoec oet aed dare hh e eos rd RS 1 10 Troubleshooting and Technical Support Lue 1 12 Chapter 2 TInstallatloti ssec eophEPICRESRATWES E RU e np eae 2 1 Connect the Hardware 0 nunun cence ees 2 3 Connecting to a Macintosh 0 00 cece eee eee eee 2 3 Computer System Requirements 0 0 00 c cece eee eee 2 6 Macintosh Computer coda 05242829440 petenexdseeeed nisi 2 6 Windows Compatible Computer 0 0 00 000088 2 7 Install Sort SL TO 4054754 i0 dE ERE AIEO a bebe IHEAEEEPEE Dad E Rd 2 8 What Is nig aNd 20320
482. ompound Source Beckman 96 2 3 mL default Compound Transfer None Triturate Not Used default Pipette Tips Layout Full rack default Compound and Tip Columns None 10 39 Chapter 10 Tutorials Wavelengths Click the word Wavelengths on the left side of the Instrument Settings dia log box to make it the active tab A black bar will appear over the word and the right side of this dialog box will then show the specific settings for the wavelengths selection If the settings are not correct as shown click the arrow next to the number of wavelengths and choose 1 from the drop down list then click the arrow next to the wavelength shown and choose the appropri ate value s for the FlexStation see Table 10 2 on page 10 39 The figure below shows how the Instrument Settings dialog box should appear at this point gm Instrument Settings x Q e vo Endpoint Kinetic Spectrum Well Scan FLEX Options Wavelengths Ex Em Auto Cutoff On 25 485 525 515 Number of Wavelengths 1 wp Sensitivity Readings 3 Excitation Emission IV Auto Cutoff oe int fs Jv Es e 55 Timing Time 200 secs i Interval 2 secs Reads 101 Minimum Interval 1 28 secs Automix Before Off Between Off AutoCalibrate Once Assay Plate Type 96 Well Standard clrbtm Wells To Read Read area amp 1 H10 Compound Source Beckman 96 2 3mL Figure 10 44 Instrument Settings Dialog Box Showing Wavelengths Settings
483. on choose a com mand from one of the drop down menus in the menu bar or press a key stroke combination to activate a command or function The Plate section is divided into four areas e Tool bar e Data display shown in a microplate grid format e Instrument settings shown to the right of or below the data display depending on the plate type and printer settings e Reduction settings shown to the right of or below the data display depend ing on the plate type and printer settings e Fluid transfer and checksum status for the FlexStation instrument When the grid in the Plate section shows colors this means that a template has been defined for this Plate section Each group defined in the template has a different color the icon of the corresponding group table will have the same color If your experiment requires multiple plates you can create as many Plate sec tions as needed The Plate menu contains the items shown in Table 3 2 shown with their tool bar or mouse equivalents These menu items and buttons are active only when a Plate section is active SoftMax Pro User s Manual 3 15 Chapter 3 SoftMax Pro Overview i Table 3 2 Plate Section Menu Commands and Tool Bar Buttons Menu Command Description Tool Bar Mouse Equivalent P Button Plate section icon Opens the Plate section in a new window l double click Plate Name of Plate section Opens the Section dialog box doued Instrument Setup in the Control
484. on of the problem but may not be com plete enough to help you remedy it At other times however errors that are not directly associated with SoftMax Pro but affect its operation may occur The information in this chapter is provided to assist you if an error occurs for which you do not have a solution or if you don t know the source of a prob lem This chapter first presents general information regarding problems you may encounter followed by specific types of problems grouped by kind display issues printing errors etc Within each group is general information regard ing such problems sometimes followed by a table containing specific prob lems and their suggested remedies If you encounter a problem during operation of SoftMax Pro first check to see if a table is associated with that type of problem and see if a remedy is listed there When remedies require more space than the table can allow a reference to a numbered text section will be listed in the table Refer to that text section by number beneath the table to learn more about solving the problem If you need additional assistance or if you encounter a problem with SoftMax Pro that is not listed here in the U S call Molecular Devices Technical Ser vices group at 1 800 635 5577 elsewhere call your local representative For problems with an instrument that do not relate to the use of SoftMax Pro please see the instrument manual for more information Message Boxes and Alerts
485. on tool bar Display Opens the Display dialog box Allows you to decide how the infor mation in the CuvetteSet section will be displayed Button Equivalent in the CuvetteSet section tool bar Graph During data collection and after data has been acquired for a Kinetic or Spectrum reading you can get a close up view of the plot shown in one or more cuvettes by first selecting the cuvette s and then choos ing this command This command is dimmed if the instrument set tings are for an Endpoint reading or if no well in the Plate display has been selected Button Equivalent in the CuvetteSet section tool bar d al Mask Allows you to hide certain cuvettes so that they will not be taken into account when performing analyses This command is dimmed if no data has been acquired or if no cuvettes in the set have been selected Button Equivalent in the CuvetteSet section Copy Template While a CuvetteSet section is open and active choosing this command allows you to copy the template from this CuvetteSet sec tion to the Clipboard allowing you to paste it into a different Cuvet teSet section in the same or in a different experiment or into a CuvetteSet section in another SoftMax Pro file 3 35 Chapter 3 SoftMax Pro Overview Paste Template If you have copied a template from a CuvetteSet section you can then create or activate open another CuvetteSet section and choose Paste Template In thi
486. on will be set to Show Reduced If reduced data is being displayed in the Plate section the initial view in the Well Graph will be reduced data and the button will be set to Show Raw e Scale to Data Scale to Limits button This button toggles between a view of the data within the reduction limits and a view of the entire data set Switching between these views helps to visualize how the limit settings affect data reduction If raw data is being viewed in the Plate section the display in the Well Graph is set initially to show the plot scaled to the reduction limits If reduced data is being viewed in the Plate section the Well Graph display is set initially to show the plot scaled to the data If you are viewing raw data and click the or the SealeTabata button only the display of raw data in the Well Graph will be affected the button will not affect the view of reduced data The same is true if you click this button while viewing reduced data To produce comparable scaling of both raw and reduced displays this button must be set to either Scale to Limits or Scale to Data for both raw and reduced views of the Well Graph 8 15 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments k 8 16 Graph Options button not found in Well Scan mode Opens the Well Graph Options dialog box which allows you enable or dis able connected points or plotted symbols the graph Graph Options k
487. on will provide a list of all areas you have viewed within the Help function SoftMax Pro User s Manual 1 11 Chapter 1 Welcome to SoftMax Pro 1 F e Text that appears in green with an underline contains a hypertext link to more detailed information about that item Figure 1 7 below shows some examples of hypertext links sOftmax Pro Help Be File Edit Bookmark Help Contents Search Back nison lt gt Softmax Pro Window Hypertext linked item clicking here will give more A Menu bar located at the top of the screen information about this item The SOF Tmax Pro window contains these major elements 4 Status bar located below the Menu bar The status bar is used to control the instrument and to display the current status of the instrument One or more sub windows containing collapsible sections in a report format The window allows you to scroll around and examine sections in the report The repart Is as wide as a piece of paper and shows you exactly what you will get when the report is printed Figure 1 7 Hypertext Links within SoftMax Pro for Windows Additional options are available depending upon where you are within the Help function More information about Windows Help can be found in the Microsoft Windows User s Guide that accompanies your Windows software Troubleshooting and Technical Support If you encounter a problem while using SoftMax Pro refer first to Chapter 11 Troublesho
488. oncentration and has no interfering color or compounds then using the water constant is fine The cuvette reference using the sample buffer or dilu ent must be used if there is any turbidity more than 576 organic solvent PathCheck should be accurate up to about 576 high salt concentration or interference in the 900 to 1000 nm range To determine possible color or com pound interference measure the OD at 900 and 1000 nm both measured with air reference subtract the 900 nm value from the 1000 nm value and then do the same for pure water If the delta OD for the sample differs from the delta OD for water then the cuvette reference must be used If the buffer is clear but the sample is turbid the error associated with PathCheck will not meet specification 2 5 for SpectraMax Plus or Plus 9 PathCheck should not be used with high levels of turbidity since the light scatter from turbidity cannot be subtracted out from the absorbance reading of the sample With the SpectraMax Plus or Plus choosing Read Cuvette Refer ence for the PathCheck option is different from a reference reading of a cuvette in a CuvetteSet section by clicking the button in the CuvetteSet section tool bar The cuvette reference used for Path Check calculations measurements at 900 and 1000 nm does not pro duce data that can be viewed in a CuvetteSet section and is used only with data in microplates not cuvettes PathCheck and SpectraMax 190 and 340PC
489. ons Read Mode Choices for the read mode are Endpoint Kinetic or Spectrum Available read modes depend on the type of instrument connected or selected in the Prefer ences see Table 4 1 on page 4 5 for a complete listing of available read modes by instrument To select a read mode click the appropriate section in the Instrument Settings dialog box Endpoint A single reading of the microplate taken at a single or multiple wavelength s Values reported are optical density or Transmittance if this has been selected in the Reduction dialog box The default reduced values are Lm1 or Lm2 Lm6 depending upon whether or not multiple wavelengths were chosen and Pathlength Pathlength applies to Plate sections in the Spectra Max Plus Plus 9 190 190PC 340PC 340PC 9 and VersaMax Plus only Li x Read Mode 0 d e Endpoint Kinetic Spectrum Buttons Automix amp Blanking Before Off Pre Read Plate Off AutoCalibrate On Strips Read entire plate Cancel Figure 4 4 Instrument Settings for an Endpoint Reading You can choose up to 6 wavelengths with SpectraMax or up to 2 wavelengths with VersaMax instruments choose 1 or 1 Ref with Emax Vmax UVmax or ThermoMax instruments SoftMax Pro User s Manual 4 9 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments k 4 10 Kinetic Data is collected over time with multiple readings taken at regular intervals The defa
490. ons 41 2 octactessss risida aoo Eb Prio dr P Puts wd pb qund 7 3 Plate Section Endpoint Kinetic and Spectrum Display Dialog Boxes 7 7 CuvetteSet Section Endpoint Kinetic and Spectrum Display Dialog Boxes 7 8 Plate Section for the SpectraMax Gemini or Gemini XS 004 7 10 Cuvelte5et Display ODEORIS rsss aristas t pandae o p m Rc pP se Pe arb 7 11 Resizing Cuvettes in the CuvetteSet Section 0 00 ununun 7 12 Default Displays for a Plate Section 0 0 0 eee eee ees 7 13 Default Displays for a CuvetteSet Section Endpoint Kinetic and Spec Modes m 7 14 Display Dialog Box for Kinetic Threshold Display 000 7 15 Endpoint Data Displayed as Threshold with Reduced Number 7 16 Kinetic Data Reduced Displayed as Ranged 0 e eee eee 7 16 Endpoint Data Reduced Displayed as Gray Scale 0022 eee 7 17 Graph Options Dialog eei sacadeicediteebesda Pag e qoe itda inent qd aded 7 19 Well Graph for a Single Kinetic Microplate Well Raw Data 7 20 Well Graph for a Single Kinetic Cuvette Reduced Data 7 20 Well Graph for Three Kinetic Wells of a Microplate Raw Data 7 21 Masked Wells in a Plate Section and a CuvetteSet Section 0 7 22 Reduction Dialog Boxes for Endpoint Kinetic and Spectrum Modes Plate Section 2 0 ccc cece ce cee ht hehehe hene 7 25 Reduction Dialog
491. ood discussion of curve fitting appears in Data Analysis and Quality Control of Assays A Practical Primer by R P Channing Rogers in Practical Immuno Assay edited by Wilfrid R Butt published by Marcel Dekker Inc New York 1984 When a fit is chosen the Graph section is updated to show the coefficients describing the fit when applicable These values are displayed in the legend at the bottom of the graph Figures 8 40 through 8 47 show the same graph with the different fit algorithms applied Note that the legend information changes along with the formula for the fit at the bottom of each Graph section for each different fit All plots on a graph must have the same type of fit When a curve fit is chosen the values for the coefficients of the equation A B C etc will be shown as will the square of the correlation coefficient R 2 The correlation coefficient describes how well a change in x values correlates with a change in the y values Please see Judging a Good Curve Fit on page 8 61 for more information The R 2 value should only be used for curve fits having linear axes SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Linear The linear function fits the best straight line to the data The equation for this fit has the form of Y A B X where A is the y intercept of the line and B is the slope A linear fit should be used whenever the values
492. ool bars in that experi ment will be hidden see Figure 3 8 If all sections are presently expanded the Expand command is dimmed unavailable Figure 3 8 Result of Minimizing an Experiment Tool Bars Hidden You may also open a section in a separate window Double clicking the sec tion icon in the tool bar or selecting New Window from the active section menu either Plate CuvetteSet Graph Notes or Group causes the currently active section to open as a separate window a copy of the tool bar still remains in the original experiment but the section is closed clicking the closed section places the new window behind the main window the original section remains closed Clicking on the triangle in the section tool bar closes the new window and causes the section in the experiment to open and clos ing the separate window opens the section within the experiment again All SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview of the tool bar options applicable to the section are available in the separate window except the triangle indicator Experimentz C3 euer ia Lambda at Maximum Plate 1 Spectrum Fluorezcence Excitation 350 Cutoff Mone EmStart 350 Stop T50 10 Automix OFF Calibrate On PMT Auto Reads well 6 Wavelength Combination Lrii pice Flate Blank Used Start 3350nm End 30nm RFU Min O RFU Max 20000 Flate Last Read 8 42 AM 67 30799 Figure 3 9 Separate Sec
493. oosing T in the Reduction dialog box 7 12 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments x Ea Plate F Plate 1 z 10 11 l maoa oa n osza oss foes oses ossa ozm ozs o2 ora o soo tar e 0 122 0135 0 14 165 16 01 E 0 A5 A6 Lmi 450 Endpoint 0 48 ose osaz oz 228 o2 ore 0300 ossz oae oe one atic o Display o o1s2 o201 022s 0246 0272 osoo osse 0367 oane os oss osar a e 0225 0246 0272 0300 082 0367 oce oss oae oer oes oes ite tast Reat r 0272 0200 052 0367 coe oae 0455 eser osos oes 0789 0017 osse 0267 nane oase 0498 ossz osos oses 0739 0817 osos 0957 1 0406 oss 0498 0 57 osos oses 0 739 817 osos poser 1102 1218 Wavelength Combination Lm1 Data Mode Absorbance 1 v E3 Pistes Eisma l4 V max Plate 1 Kinetic Time 5 700 Interval 0 30 11 a Lri 260 Automix Off Calibrate On Kinetic Display Lag Time 0 00 End Time 3 00 OO Min 0 OD rax 1 Wavelength Combination Lm1 Ymax Pts 11 711 Data Mode Absorbance Flate Last Eead 8 43 APT 7 16 99 s BE e ELE EZ Lambda at Maximum Plate 1 Em Start 350 Stop 75 Step 10 Spectrum amp utamix OTF Display Calibrate On Start 250nm End 7 5 nm OD rin OD rax 1 Wavelength Combination Lm1 Plate Last Read Data Mode Absorbance 6 43 AM 77 16799 Figure 7 7 Default Displays for a Plate Section SoftMax Pro User
494. or all instruments that are capable of Kinetics the default Kinetic reduction is Vmax Other reductions available from the pop up menu are Time to Vmax Onset Time Time at Minimum Time at Maximum Time at 1 2 Maximum Slope Area Under Curve or you can choose Custom and then create a different reduction formula Vmax The maximum slope of the Kinetic display of optical density versus time The default value displayed is in milli optical density units per minute mOD min The rate is calculated with the use of Vmax Points see below which determines the number of contiguous points over which Vmax is cal culated The number of Vmax Points defaults to the maximum taken during the reading you can change this number in the Reduction dialog box if desired 0989 Vmax Vmax sec e 60 000 A linear regression is performed to determine the slope of the line using the equation y m X B where m is the slope of the line The reaction rate is reported in milliOD min color change in milliOD per minute of reaction time Vmax Points This setting defines the maximum size of the line segment used to determine the slope of the line used in calculating the rate of the reaction The default is the total number of points taken in the reading with the data normalized to a starting point of 0 0 as opposed to starting from the optical density of the first time point The first slope is calculated for a line drawn beginning at the first readin
495. or choose Create Summary from the Notes menu The Calculation dialog box that appears see Figure 8 31 allows you to enter a description of the summary accept the default title for the summary change it or hide it and enter a formula including the number of decimal places the reduction should show if appropriate Detailed information regarding oper ators and accessors can be found in the Formula Reference Guide If you wish to see the formulas associated with the summaries without calling up the Calculation dialog box for each one you can choose Show Formulas from the Notes menu To see these formulas only briefly hold down the shift and control Ctrl keys while that Notes section is active The summaries will expand to show the complete formula along with the other summary infor mation They will resume their previous appearance when you release the option key Graph Section The Plot X Dialog Box SoftMax Pro User s Manual Whenever you create a new Graph section or add a plot to an existing graph the Plot X dialog box opens automatically X starts with 1 and incre ments each time a new plot is added to a Graph section In this dialog box you can rename the plot assign specific information to be plotted on the X and Y axes and choose which icon and color will be used If you rename the plot the title of the dialog box will change to show the new plot name All groups that have been created or assign
496. or one or more sections within an experiment will cause the print setting button for the experiment to change to reflect an undecided status with regard to the sections within it Toggling the experiment s print setting button will override any changes made to individual sections and set all of them to print ing When Print is chosen from the File menu selecting Print All will override the print settings made to sections or experiments Customizing the Report In addition to choosing which sections will print in the report by toggling their print status you can choose to print all items in a file or to print only the report from the Print dialog box that appears when you choose Print from the File menu 3 6 copies 1 LI Collated Pages amp All Paper Source amp All pages from Auto Select First page from Cassette 250 sheets Remaining from Cassette 250 sheets Figure 9 7 Print Dialog Box Other options regarding the appearance of the printed report can be chosen in this dialog box you can add text to be included in the header on every page SoftMax Pro User s Manual Chapter 9 File Management and Printing of the report maximum of 100 characters in a single row and you can include or exclude the experiment title the name of the experiment in the file as well as the name s of sections and you can include the date and time of printin
497. or the Life Science and Drug Discovery editions of SoftMax Pro remain unchanged PPR and PDA New file extensions are created by Enterprise Edition software however EDA data and EPR protocol These new file extensions will be recognized only by Enterprise Edition software prohibiting opening or modification of such files by other editions of SoftMax Pro This helps to ensure that the integrity of the 21 CFR Part 11 compliant features of the Enterprise Edition is maintained The Enterprise Edition can open all file extensions from all other versions User access to the Enterprise Edition of SoftMax Pro is tied to a database cre ated and saved with Enterprise Administrator User privileges are set within Enterprise Administrator and these privileges determine which menu choices are available to that user within SoftMax Pro A user may also log in to the Enterprise Edition as a guest That user will be able to open view files but may not make any modifications to the documents and may not save them under the same name Chapter 1 Welcome to SoftMax Pro 1 F About the Documentation The SoftMax Pro documentation set consists of this manual which includes written tutorials an on line help system and a formula reference guide Additional documents on disk may also be provided to cover the latest infor mation regarding the software An outline of the contents of this manual fol lows along with introductory information that should
498. orbance of the sample If no reference reading is taken the SpectraMax Plus or Plus 9 will use the Ig values stored in the NVRAM of the instrument A reference reading of the cuvette in the SpectraMax Plus or Plus can be made at any time by choosing Reference from the Control menu or from the CuvetteSet section status bar The Reference button is available when the active section is a CuvetteSet section It is not visible when the active section is not a CuvetteSet section or if multiple sections are selected If no reference has been taken in a CuvetteSet section and you then read a ref erence SoftMax Pro assumes that this reference will be used with all cuvettes in the CuvetteSet section Therefore the time and date of the reading are dis played under each cuvette in the section three cuvettes per row and one cuvette per row displays only The reference will also be applied to any new cuvettes created in that section The reference may be read before or after reading samples You may have more than one reference per CuvetteSet section If you have a single cuvette to which you want to apply a different reference highlight that cuvette in the CuvetteSet section place the second reference in the cuvette 4 41 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 1 k 4 42 port and click Ref The time date stamp of the reference for that cuvette and only that cuvette in the set will change NOJ An
499. ore than one Plate section and no Plate section is active choosing the Read command will cause a dialog box to appear asking you to choose which section to read see Figure 5 33 Select the correct section and SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments then click to close the dialog box If a Plate section is active choosing the Read command will read that plate Select Section Fal Sections Figure 5 33 Select Section Dialog Box If you select a Plate section that already contains data and choose to Read again an alert appears asking you to confirm replacing data in the destina tion plate see Figure 5 34 If you select Replace the reading will proceed and the previous data will be replaced with new data Replace data in Plate 1 Cancel Replace Figure 5 34 Alert Regarding Replacing Data for Plate Section General Information about a Reading After beginning a reading the status bar will show the actions that are occur ring in the instrument along with a box showing the elapsed time for each action in minutes and seconds The button will change to Stop allowing you to terminate a reading if necessary An example of the appear ance of the status bar at the start of a Kinetic reading of a microplate is shown below Plate Reading 7 af 4 U o n0 04 Figure 5 35 Status Bar during a Kinetic Reading SpectraMax Column vs Wav
500. ormation Printshop from Broderbund uses the pda extension to identify its files as does SoftMax Pro See General Problem 2 below for more information On the PC SoftMax Pro limits file names to 59 characters plus a period and a 3 letter extension for a total of 63 characters It is possible to make a file name longer than this by manually changing it in a direc tory listing or by using the Save As command in the software A file with a name longer than this limit will no longer be able to be read by SoftMax Pro may not show up on file lists and will return an error if you attempt to open it File names that are made too long using the Save As command will show corrupted characters such as y etc for any letters that fall after the 63rd 1 The amount of memory in the computer may not be sufficient or the operating system may not be optimum Check the amount of RAM and the operating system version to see if they meet the minimum requirements outlined in Chapter 2 Installation 2 SoftMax Pro may be trying unsuccessfullyto communicate with the printer port If a printer is connected check that the computer can see and send data to the printer If a printer is not connected Soft Max Pro still requires that a printer driver be installed choose any printer 3 SoftMax Pro may be trying unsuccessfully to communicate with a serial port a Another device such as a camera a handheld PDA device or another i
501. ort a tab delimited time tagged file from FlexSta tion data SoftMax Pro is also able to re import the time tagged file data e A tab delimited text file with time interpolated data can be exported from SoftMax Pro Such data can facilitate analysis using third party software such as Microsoft Excel Time interpolated files cannot be re imported into SoftMax Pro e The import feature has been expanded to include the ability to import a tab delimited text file exported from FLIPR Autoread e This new feature is included for all instruments not available in Kinetic and FLEX read modes Autoread enables the sequential reading of multi ple plates within an experiment You can set intervals between plate reads if desired Reduction Modifications e A new Kinetic reduction dialog box is available for FlexStation read mode that adds special display options Raw Values Baseline Offset Values and Increase Values These display options are used to alter the way in which the data is graphed Additionally for Slope and Area Under the Curve cal culations for curve fitting the raw data is smoothed using a simple moving average You can choose the period of this moving average from a popup menu e Additional reduction formulas are available in the FlexStation Kinetic reduction dialog box Time at Minimum Time at Maximum Time at 1 2 Minimum Peak and Max Min A 6 SoftMax Pro User s Manual Appendix A SoftMax Pro Version Specific
502. orts the time at the minimum RFU that falls within the reduc tion limits Time at Maximum This setting reports the time at the maximum RFU that falls within the reduc tion limits Time at 1 2 Maximum This setting reports the time at the half of the maximum RFU that falls within the reduction limits To calculate this reduction SoftMax Pro first determines the point within the reduction limits that has the maximum signal level RFU It then scans the plot from left to right until it finds two points having signals that bracket half of that value A linear interpolation between these two points is used to estimate the Time at 1 2 Maximum SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual Peak This setting reports the maximum RFU value It will be adjusted for different smoothing options Max Min This setting represents the difference between the maximum and minimum RFU values If the minimum RFU value is below zero it will be adjusted so that the minimum is always zero Slope This setting determines the slope of the combined plot i e the slope of the line using linear regression after the wavelength combination reduction This setting uses all visible timepoints in the reduction window Slope is the same as Vmax Rate when Vmax Rate is set to the same number of points as the run i e the default Slope is not the same a
503. otes Section Edit Summary New Window Export Graph Section Name New Window Display ne CALES Copy Template Show Formulas Export Template Import Template Group Settings New Window Section Name Window Macintosh Help Windows Help New Window Tile Hep Cascade About Balloon Help Contents Arrange Icons Show Balloons Index SOFTmax PRO Help Using Help Formula Help About SOFImax Pro Figure 3 16 SoftMax Pro Menus w 1 Untitled SoftMax Pro User s Manual 3 25 Chapter 3 SoftMax Pro Overview Apple Menu About OFTmax PRO File Menu Mew Open Clase Save Save As Import Export Set Password Page Setup Print Standard Curve Print aeP Quit ae 3 26 About SoftMax Pro Macintosh only Displays the SoftMax Pro version registration information ROM version if available from the instrument and copyright information If an instrument is connected to the Macintosh running SoftMax Pro the instru ment type will also be displayed You will need to know this information if you call for technical support Other items in the Apple menu depend on the settings of your Macintosh New 8 N CTRL N Opens a new file window allowing you to begin a new or different data file Information from each SoftMax Pro window which can consist of one or more experiments wi
504. oting and try to locate a remedy for the problem For problems with SoftMax Pro that are not listed there or if you need additional assistance in the U S call Molecular Devices Technical Services group at 800 635 5577 elsewhere call your local representative 1 12 SoftMax Pro User s Manual Chapter 2 Installation Connect the Hardware eser RE Sud we pDPHEFUIVE VIC EO 2s 2 3 Connecting to a Macintosh e c oe d ive i onde dee 2 3 Computer System Requirements eee eese 2 6 Macmtoshk Computer co o oux dde Sind etie tap E Rd aub 2 6 Windows Compatible Computer 0000000005 2 7 Install SoftN ax DEO i wsbak a abs goals aati bn hia ese podem Oo ud is 2 8 What Is Installed uus s i6 ith EUER EH rier as Ehe pt 2 9 Enter Registration Information sssseeeeeeeee eee ee 2 13 Communicating with the MDC Microplate System Instrument 2 15 DGET TeIerellGes uoo sd oes EMEN PE e si ata m ent 2 16 Serial Poll acta Susu k ewe ESQ ONE EDEN E UNES cn 2 16 EXDOEPU TOEDUOU 663434 2200 23 nde TRU ee aUe iS e ti ede vas 2 17 Reddetooreseqe e ved diuo Uere db eut atate ute edt iu 2 18 PIT Grech os ER Ero pU REA SII de dl DES a eects Resa 2 19 INULOSAV CG os EESE wes rti lippi indie n 2 20 PULOP ING 23295 pee Sc OUS Road Pod v an p a d o 2 22 PSSA vs Foldefc is soo prv aee ENDURO r LI wir ay vd mb 2 23 Chapter 2 Installation 1 k NOH Enterprise Administrator must be installed by an administrator Detailed insta
505. otomultiplier tube Gray Scale Data Display Raw or reduced data is displayed proportionally in a plate format using seven shades of gray ranging from light shading at the low end to dark shad ing at the high end based on user defined high and low limits Group Wells can be assigned to group types using the Template Editor Depending on the default protocol used certain group types such as Standard or Unknown may be created automatically you can create others as required Highlight Selecting an object or piece of text is selected so that it will be affected by your next action Highlighted text appears in reverse video or in color Highlighted objects may change color or be surrounded by a selection cursor Icon A graphical representation of an element of the computer or software such as a disk drive application or file Import Bringing information or data from another application into SoftMax Pro Information can be brought into SoftMax Pro in two ways by copying tab delimited text to the clipboard and then pasting this information into the Plate section or CuvetteSet section within SoftMax Pro or by using the Import command under the File menu to import ASCII text files having spe cific formats SoftMax Pro User s Manual B 7 Appendix B Glossary of Terms 9 a Incubator Choosing Incubator from the Control menu or clicking the incubator button opens a
506. ou wish to enter or you want the concentration or dilution factor to remain constant This can be done using the button setting the starting value to 1 or another constant value and either multiply ing by 1 or dividing by 1 In this way the names will increment but the value of concentration or dilution will not change If you don t have a concentration or dilution factor simply deselect the sample descriptor checkbox if it is selected If sample names are set to increment automatically using the Series function be aware that SoftMax Pro will automatically truncate the sample name to three or four characters including the incrementing number if the sample name starts with letters If the sample name is entirely numeric it will not be truncated If sample names are very long and they must be kept long for reference you will have to set up your replicates manually and will not be able to use the series function Blanking Two types of blanking are available in SoftMax Pro for fluorescence instru ments Understanding the differences between the types of blanking func tions will enable you to choose the type of blanking that is appropriate for your assay Note that blanks can be used in combination and have cumulative effects The types of blanking that are defined in SoftMax Pro are 5 30 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments e Plate blank e Group blank Plate Blanks
507. oups in the Template Editor In this tutorial some groups were already cre ated when you started the new file and a new one was added when you cre ated the new group Sam w dil The content of Group sections changes depending on the wells that are assigned to these groups in the Template Edi tor Before a plate is read these sections show no data for the wells After a reading Group sections contain information about the values for the wells contained within the groups Scroll down until the Standards Group is visible It should look similar to this TI standards us 24 cfe aj Standards paqimi Figure 10 28 The Group Section for Standards Absorbance shown The columns in this group are set up as follows from left to right e Sample the seven sample names assigned for the Standards group Concentration the concentrations assigned to each sample in the template 10 25 Chapter 10 Tutorials 10 26 A B e BackCalcConc Returns the curve fit associated with the plot of Standards from the Standard Curve against the Values column e Values raw OD values obtained for each sample e MeanValue the mean of data from the Value column for each sample e StdDev standard deviation for each sample value e CV coefficient variation percent for each sample value In the following part of this tutorial you will view formulas associated with columns and summaries resize individual columns autosize all the co
508. ow The maximum positive or negative slope is reported as Vmax RFU sec or RLU sec Vmax Points can be set from 2 to the total number of points in the curve The number of Vmax Points can be set before the plate is read and can be changed after the plate is read The data display will update automatically each time The Vmax Points setting does not affect data collec tion The height of the Y axis in the raw data plots or plate display is con trolled using MinRFU RLU and MaxRFU RLU The default settings are MinRFU RLU 0 MaxRFU RLU 20 000 The width of the X axis in the raw data plots or plate display is con trolled using Lag Time and End Time If either the MaxRFU RLU or End Time setting excludes points such that a total number of readings equal to Vmax Points cannot be used then the maximum included points will be used Time to Vmax This is an alternative method for analyzing non linear Kinetic readings that reports the elapsed time until the maximum reaction rate is reached rather than reporting the maximum rate itself Used in conjunction with Vmax Points Time to Vmax is the time to the midpoint of the line defined by Vmax Points and used to calculate Vmax This elapsed time data is useful for appli cations including coagulation chemistry where the changing concentration of the reagents does not change Vmax but rather will change the time at which the reaction reaches the maximum rate 6 27 Chapter 8 Displaying
509. p elc d bp dd EN Ra Es eS A 3 SoftMax Pro 3 1 Update Specification 0 0 00008 A 3 SpectraMax Plus384 support 0 0 eee eee A 3 SpectraMax Gemini XS support 0 0 0 eee eee A 3 Well Scanning Read Mode 0 2 0 cece eee eee A 3 Apple OS 9 Navigation Services 00000 e eee eee A 5 Interprocess Messaging i42 4 4 Far baer do dedo d na beanie A 5 Instrument Settings Changes 00 eee ee eee A 5 Password Protection oce 67 ead aintneen to tub aridi ep aw ene A 5 Minimum Kinetic Interval 0 00 0 00 ee eee eee A 5 Autosave Changes 0 0 0 e cece eee eee eens A 5 Autoprint Changes ouieo redo riste rs bir eos S eaten seme A 5 SoftMax Pro 3 1 1 Update Specification 000005 A 6 Correction of Problems in SoftMax Pro 3 1 Lus A 6 New Interprocess Messaging Features 0 A 6 SoftMax Pro 3 1 2 Update Specification 000008 A 7 New and Revised Interprocess Messaging Features A 7 SoftMax Pro 3 2 Update Specification 0 00 00 0 008 A 7 FlexStation Features orien urpis niekes Up date hae bomen A 7 Now JACCOBSOIS des ume SiS ba te d RO tnde d e A 8 CIBC SUPE eiconsectet eror etobpr adeste rad p ad eiie A 8 porn POOL 222 cecaccnedsastea oman tran e eA A 8 vd sca sensed oe ener eat anes nee eee hoes A 8 Reduction Modifications 0 00 c eee eee eee A 8 SoftMax Pro 4 0 4 01
510. pectra Max Plus or Plus 94 one involves cuvette measurements cuvette reference and the other involves a pathlength correction constant stored in the instru ment water constant Pathlength correction for the SpectraMax 190 and 340PC uses the water constant The actual pathlength d of a solvent is found from the following equation Sample OD io OP a0 k d cm When a cuvette reference is used for pathlength correction the value of k is obtained by taking OD measurements on the fluid in the cuvette at two wave lengths 1000 and 900 nm k Cuvette OD 000 ODo00 When using the pathlength correction constant water constant the value of k is obtained from the instrument This constant is recorded into nonvolatile SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments memory when the instrument is manufactured and may differ slightly from instrument to instrument Once the actual pathlength d in cm is found the following equation is used to perform the pathlength correction lcm x lcm d cm OD OD sample Pre Read Blanking vs Background Constant Subtraction SoftMax Pro User s Manual In previous versions of SoftMax Pro selecting PathCheck automatically enabled the Pre Read Plate blanking function which is used to subtract the absorbance due to the plate material on a well by well basis before path length correction is applied Now however
511. pened The second plot name defaults to Plot 1 which now shows beneath the first plot Change this second plot name to Standard 2 choose Standard2 for the group and change the X and Y axes to show concentration and values respectively for Standard2 Graph Options Title Graph Height 100 Type onts s Sample Text Tren we O E Sample Text lil QQ Stack bar Legend Connect points Sample Text Axes ES Plot symbols al Q Standard 1 O Standard 5 Doncentrationimatandardz Y MaluesitStandard A Errorz Wo Error Y Errors Mo Error cancer Foe Figure 8 36 Graph Options Dialog Box with Second Plot Added for Standard 2 5 This process is repeated for a third plot labeled Standard 3 SoftMax Pro User s Manual 6 49 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 6 Now that all plots information is complete clicking in the Graph Option dialog box will close it and the resulting graph will show the three plots created XT 47 Graphite ite Fit Logoa w s 5 Graphz 0 93 0 83 0 73 0 63 0 53 0 43 0 33 0 23 0 13 Concentration OS D ex AX BD A Plaot 1 Standarde1 concentration ve Means alue O 416 2 Platz3 Standards Concentration ve Means alue 0 62 O Plot 2 Standards2 Concentration vs Mean s alue 0 925 Figure 8 37 Graph Displaying Three Standa
512. pening the Calculation dialog box for each one you can choose Show Formulas from the Group menu To see these formulas only briefly hold down the shift and control Ctrl keys while that Group section is active The summaries will expand to show the complete formula along with the title They will resume their previous appearance when you release the option key Summaries in Group sections can be moved horizontally if desired by drag ging them left or right When you position the pointer over a Group sum mary the cursor changes to show a double headed horizontal arrow Clicking once highlights the summary dragging and then releasing the summary leaves it in the position where the mouse button was released To have summaries line up with particular columns in the Group hold down the control Ctrl key while dragging the summary When you release the con trol Ctrl key the summary will snap back so that it begins flush to the left side of the column under which it was released Each summary can be posi tioned in a different place in the Group section if desired SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments NOH To delete a summary highlight it and press the Delete key or choose Cut from the Edit menu A summary can also be copied or cut and then pasted in a different location You can also change the format of the text in Group section columns and or summaries By selecting
513. pens the Well Graph Axis Settings dialog box for the particular axis allowing you to set Auto Range parameters choose the minimum and max imum values for the selected axis cause the axis to be auto ranged add or remove gridlines and add or remove axis tick marks When a Well Graph is opened the default settings are to show the reduction limits for that axis with Auto Ranging off and gridlines and tick marks selected Only the Well Graph being viewed is affected by changes in these dialog boxes Done Closes the Well Graph dialog box SoftMax Pro User s Manual 7 19 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments i M Plate 1 Well Graph Raw 150 Time secs Yroax Points 34 Well oO A3 max 237 4 R Z 0 310 e JL J Done Jr Figure 7 14 Well Graph for a Single Kinetic Microplate Well Raw Data OD z Puvettesetz1 well Graph Reduced p EH 150 Time secs Yroax Points 24 Cuvette O A max 167 76 R z L5 J Ls Jes nene Jr Figure 7 15 Well Graph for a Single Kinetic Cuvette Reduced Data Multiple wells from the same Plate section or cuvettes from the same Cuvette Set section can be graphed together in one enlarged window Select the well s or cuvette s in the data display hold the Shift key if you are selecting non contiguous wells or cuvettes you can drag across contiguous wells and then click the Graph button or double click in one of the selected wells or cuvettes 7 20
514. plate and Group Graph and Notes sections However depending upon the particular assay and how you wish to report the data only some of these sections may be or need to be present in every file For example some simple assays allow you to calculate concentration in the Plate sections In such cases Group and Graph sections are necessary only if you wish to display the data graphically The following diagram shows the relationship between the different sections Standard Group Table Reduced Number from Plate in SoftMax Pro Reported Here Plate with Graph Section Template Plots Created with Initial Data from Information from Unknowns Microplate C IS Standards Reader P Group Table Reduced Number from Plate Reported Here Interpolated Values Results Reported from Graph Section Figure 8 1 Relationship of Sections This chapter discusses the initial display and reduction of data in the Plate section first followed by display and data analysis in the Group Graph and Notes sections Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 1 k Initial Data Display and Reduction The manner in which data is displayed in a Plate section depends on the type of reading that was performed Endpoint Kinetic Spectrum Well Scan or FLEX but can be customized by choices made in the data display and reduc tion settings Depending on the type of reading perfor
515. plates were not defaulting to the correct plate type e Wavelength settings for Gemini Spectrum reads were not being correctly verified Problems occurred with Start wavelengths higher than Stop wave lengths e The wrong error message appeared when the number of Kinetic reads was set higher than 10 000 e AutoPMT was available in Kinetic mode on a fluorescence reader when an absorbance Kinetic protocol was opened Also AutoPMT was available on the Gemini for spectra when an absorbance Spectrum file was opened New Interprocess Messaging Features The NewPlate command creates a new Plate section in the currently selected Experiment The plate will be named following the normal rules for SoftMax Pro This plate name will be Plate X where X is the lowest number not already being used for a section The NewNotes command creates a new Notes section in the currently selected Experiment The notes will be named following the normal rules for SoftMax Pro This notes name will be Notes X where X is the lowest number not already being used for a section The SaveAs OpenAssay ExportAs and ImportTemplate commands have been modified to allow full pathnames The file path can be fully redirected by beginning the filename with a partition letter and a colon D V mySubDi rectory myFile for example A 6 SoftMax Pro User s Manual Appendix A SoftMax Pro Version Specifications SoftMax Pro 3 1 2 Update Specification New and Revis
516. plied With simulated data apply a linear fit by clicking in the menu next to Fit and choosing Linear Releasing the mouse button causes SoftMax Pro to redraw the graph based on its evaluation of the fit of the data to the new curve choice The figures below show a linear fit followed by a 4 parameter fit Mean Yalue 0 22 Graph 1 LENT LS STL LLL LTTE TLE TELE MPDCHOMATNIESPI Leen eee 0 12 T 0 0 2 0 4 0 6 0 8 Concentration y A Bx A 0 133 Std Standards Concentration vs Mean alue x 2 Grapnitt E Fit 4 Parameter_ Standard Curve myg EE Sees mes z o0 3 4 gt c Dd D H H 0 22 AE ad 0 12 0 1 1 10 Concentration y CCA DW GUC B D A B c D R 2 Std Standards Concentration vs Mean value 0 132 0 993 111 97 23 269 0 998 Figure 10 39 Standard Curve Graph Section with Linear and 4 Parameter Fits Applied 10 32 SoftMax Pro User s Manual Chapter 10 Tutorials SoftMax Pro User s Manual You can also display the data in a bar graph or another format by clicking the graph options button 2 which opens a dialog box like that shown in the fol lowing figure W Graph Options x Height E onts Sf Scatter Tile Sample Text Mh C Cluster bar sample Text Connect points aves Sample Text S
517. plots format raw OD values with respect to time or wavelength While a Kinetic or Spectrum reading is in progress the display updates dynamically to show the data collected so far If the instrument is reading more than one wavelength in Kinetic mode plots for the different wavelengths are overlaid in each well All choices except those defined in the Instrument Settings affect only the manner in which the data is displayed the raw data is not changed permit ting you to optimize the display after data is collected Data reduction changes the way in which data is displayed and also affects the values used in calculations within SoftMax Pro While the raw values remain unchanged calculations will use reduced values if any and are therefore affected by settings chosen in the Reduction dialog box For more information see Data Reduction on page 7 23 With the SpectraMax Plus Plus 190 340PC and the VersaMax data comes from the instrument to SoftMax Pro with four digits to the right of the decimal point instead of three digits for all other instru ments Only three of the four digits are displayed in the Plate section or CuvetteSet section for Endpoint data however the fourth digit is rounded up or down 0 0045 rounds up to 0 005 0 0044 rounds down to 0 004 for example You can view all four digits of data if desired by displaying the raw OD in a Group section table and increasing the number of decimal places showing
518. possible interval for Kinetic readings using SpectraMax and VersaMax instruments choose wavelengths in ascending order Spectrum SpectraMax instruments only Measures absorbance optical density or Transmittance across a Spectrum of wavelengths The default reduced value reported for each well is the wavelength of maximum absorbance when opti cal density is displayed or the wavelength of minimum absorbance when Transmittance is shown gm Instrument Settings x O ie pe Endpoint Kinetic Spectrum Options Wavelengths Start 350 Stop 750 ee es Start nm Pre Read Plate Off Step nm AutoCalibrate PE ENEON EEEE On Strips Read entire plate AutoRead Off Speed Read Ts Cancel Figure 4 6 Instrument Settings for a Spectrum Reading All Spectrum readings are made using the scanning monochromator of the SpectraMax Wavelengths For Endpoint or Kinetic Readings SpectraMax Instruments Choose the number of different wavelengths to be read At least one wave length must always be specified Selecting more than one wavelength will cause a corresponding number of wavelength setting boxes to appear maxi mum six wavelengths SoftMax Pro User s Manual 4 11 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments imi 280__J w tma eso_ Lm2 aos e PINE Figure 4 7 Wavelength Settings for SpectraMax Instruments The wavelengths specified previously or the defau
519. printer Place a note on the top of the switch box indicating the identity of each output port SoftMax Pro User s Manual Chapter 11 Troubleshooting 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 To Switch from Reading to Printing Configure the computer SoftMax Pro and the switch box as described above for reading a microplate Read the microplate Set the switch box to communicate with the printer Quit SoftMax Pro In the Finder select Chooser from the Apple menu to bring up the Chooser dia log Set AppleTalk to active In the box on the left side of the Chooser dialog choose the AppleTalk driver for the printer The printer name should appear in the box on the right side of Chooser dialog Close the Chooser dialog Start SoftMax Pro Print the desired sections To Switch from Printing to Reading Configure the computer SoftMax Pro and the switch box as described above for printing Print the desired sections Select Chooser from the Apple menu to bring up the Chooser dialog Set AppleTalk to Inactive Set the switch box to communicate with the instrument Select Preferences from the Edit menu Without changing any settings click OK Read the microplate SoftMax Pro User s Manual 11 13 Chapter 11 Troubleshooting Display Problems A large Group section table You are attempting to display more than 2500 data points in a group becomes gray or
520. ptimize the display after data is collected Data reduction changes the way in which data is displayed and also affects the values used in calculations within SoftMax Pro While the raw values remain unchanged calculations will use reduced values if any and are therefore affected by settings chosen in the Reduction dialog box For more information see Data Reduction on page 8 19 Data comes from the instrument to SoftMax Pro with four digits to the right of the decimal point Only three of the four digits are dis played in the Plate section for Endpoint data however the fourth digit is rounded up or down 0 0045 rounds up to 0 005 0 0044 rounds down to 0 004 for example You can view all four digits of data if desired by displaying the raw values in a Group section table and increasing the number of decimal places showing 8 5 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 1 k 8 6 The Display Dialog Box Before or after a plate has been read you can click the Di spl ay button to open the Display dialog box which allows you to change the way the data is presented Choices available in this dialog box depend on whether a Plate sec tion is currently active and which read mode you have chosen for example the choice for Plots is not shown if Endpoint mode is selected Figure 8 2 shows the default Display dialog boxes for the different read modes The Display dialog box is
521. puting by William H Press Brian P Flannery Saul A Teukolski and William T Vetterling published by Cambridge University Press New York 1988 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Point to Point A linear equation is fit to each pair of data points Fit parameters are not given The point to point curve fit is a linear fit composed of pieces that assume a linear relationship between each pair of data points The line seg ment defined by each pair is used to interpolate data between those points Since there are multiple line segments fit parameters are not shown on the graph v 2 Graphits Fit E Graphz3 B Concentration Ploti Groupe2 Concentration vs values Figure 7 49 Graph with Point to Point Fit SoftMax Pro User s Manual 7 61 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Cubic Spline This curve fit generates a fit to a cubic equation between each pair of data points The general form of a cubic equation is y A Bx Cx Dx The equations are computed with the requirement that the first and second derivatives of the equations are continuous throughout the range of the data As multiple curve fits are performed with this routine fit parameters are not shown with the graph x Graphs Fit zi Graphz4 B concentration Plot 1 Groupzz Concentration vs Mean alue Figure
522. quent data set in the file includes the lines described above a blank line and a line with the termination field Importing Data from Analyst Acquest ScreenStation and FLIPR SoftMax Pro imports data in tab delimited ASCII text format allowing you to import data collected by the Analyst Acquest ScreenStation or FLIPR instru ments into SoftMax Pro Note that only data that can be displayed in Plate and or CuvetteSet sections can be imported Three choices are available for importing information into SoftMax Pro 4 1 Import e Import allows you to bring data from a run that was saved previously Export using a MDC Microplate System instrument e Import Analyst allows you to import verbose reports generated using Cri Import Criterion Import FLIPR terion Host or SCREENPLAY for all instrument modes e Import FLIPR allows you to import FPD files exported from FLIPR software The Tutorial folder contains examples of exported data from these instru ments and the Assays menu lists protocols that are specific to these instru ments Import Selecting Import from the Import Export menu under File causes the Import dialog box to appear SoftMax Pro User s Manual 9 21 Chapter 9 File Management and Printing L3 Data Files Avalon Plate Cuvette Sections Eject Raw Data w settings Spectrums multiple Cancel Figure 9 15 Import Di
523. r columns you select depending on the filling direction the remaining wells that cannot contain replicates will be labeled as additional individual wells in the series For example if you fill a block of 4 x 4 wells from the top and choose three replicates then the first three columns will contain the replicates and the last col umn will contain the additional wells in the series see the last diagram in Figure 5 29 This replicate feature allows you to quickly define the number of replicates and in cases where the series has more samples than a single row or column to name them simultaneously To create a series with all 96 samples highlight all the wells in the plate select the fill direction and set the replicates to 1 5 29 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Fill from Top one replicate Fill from Bottom one replicate Fill from Left two replicates Fill from Right four replicates Fill from Top three replicates Figure 5 29 Fill Options and Replicate Settings 7 Click OK to save the series and close the dialog box While still in the Template Editor you can view the value assigned to any well by click ing that well The value will be displayed at the top of the Template Ed itor Holding down the Ctrl Shift keys will display the values for all wells Sometimes you may wish the names of a series of samples to incre ment but may not have a concentration or dilution factor that y
524. r list John Smith will na longer be allowed to use any software Are you sure you want to remove John Smith oma Figure 6 5 Remove User Warning Message If the user really should be removed click the button The removal of a user is immediate and that user will disappear from the Users list box in the Users property page Change Password The administrator may change the user s password This step doesn t require entering the old password The administrator may simply assign a new pass word as shown below IITUTEENENENNRRM M 4 New Password LLLE EIE Confirm Password ITTIITI ca Figure 6 6 New Password Dialog Box The new password must be at least six characters long and must contain at least one numeric and one alphabetic character Global options appear in the lower left corner of the Users property page Password Aging and Idle Time out These options are disabled if their check boxes are empty If checked the administrator must enter either the duration of password aging or the desired idle time out period Global Options Pane Global Options Password Aging o Months Idle Time out 0 Minutes Lock software after logon failures Figure 6 7 Global Options Pane SoftMax Pro User s Manual Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator SoftMax Pro User s Manual Password Aging indicates the period of time in months after which user
525. r previous versions of SoftMax Pro e Appendix B Glossary of Terms provides a list of terms used in this man ual and their meanings This manual assumes that you are familiar with the basic operating environ ment of your computer We recommend that you become familiar with your computer s operating features prior to installing and using SoftMax Pro Conventions Used in This Manual This manual uses visual cues and standard text formats as follows Visual Cues The following typographic conventions are used in this manual Format Menu Item Text Entry Key Name Meaning Menu titles and selections Keys that you press on the keyboard or placeholders for information you must provide entries that you type from the keyboard Information shown in this font should be entered exactly as it appears For exam ple if you are asked to type filename you would type the actual name for the file instead of the word shown in this font Buttons shown on the screen Note that sometimes an actual representation of the button may be shown The accompanying text applies only to SoftMax Pro for Macintosh The accompanying text applies only to SoftMax Pro for Windows Keyboard Formats Key designations combinations and sequences appear in the following formats Format Meaning 3 Key Selections on the Macintosh can be made using the mouse but often shortcuts are available using the keyboard Shortcuts com monly use the comma
526. racted from the optical density of all the other cuvettes in the CuvetteSet section Procedure 2 1 2 3 4 Create a cuvette set with as many cuvettes as you wish up to the maximum of 96 Reference and read the cuvette Create a template with the appropriate groups Create a column in each Group table with the formula wellpreread Lmi If you have a multiple wavelength reading and wish to see the ref erence at each wavelength create additional columns with the for mula wellprereadLx where x is 2 3 or 4 corresponding to the wavelength for which you want to see a reference The optical density with the reference subtracted will be displayed in the Val ues column The reference value for each sample will be displayed in the cus tom column you have created SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Reading a Cuvette You can highlight any cuvette in the CuvetteSet section and choose to read it If you select more than one cuvette the software looks for the leftmost selected cuvette that does not contain sample read data When one is found the reading begins with that cuvette If you select a cuvette that already contains data and choose to Read again an alert appears asking you to confirm replacing data in the destination sample see left side Figure 4 40 Similarly if you choose Reference while a sample that has already had a reference readi
527. raph window if desired by clicking the Print button at the bottom left corner of the dialog box If you enlarge the Well Graph to greater than 12 inches the graph will not fit on an 8 5 by 11 inch page and the printout will be trun cated SoftMax Pro User s Manual 7 21 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Masking Wells or Cuvettes If you find data in certain wells or cuvettes outliers that should be excluded from the calculations to be performed by data reduction you can mask these wells or cuvettes Select the well s or cuvette s to be masked and then click the Mask button in the Plate section or CuvetteSet section tool bar or choose Mask from the Plate or CuvetteSet menu Masked wells cuvettes are shown with an overlay of diagonal lines The masking function can be used as a what if tool For example you could suppress or enable certain functional groups within the experiment Suppose you have included a group blank in the template and want to see the data with and without the blank Masking the group blank wells would sup press the blanking function unmasking them would enable it again Plate 1 2 050 0 020 sors ones onsz oon oos ERE B 0 008 0 005 0 052 0071 0 092 p 0142 0 170 020 m E 0 085 H on 7 uos Automix Off Calibrate On Flate Last Read 19 52 AM 7 16 23 4 0 365 0417 0475 0538 e RA eee pes 2 3 ll Masked lb hl ll IL Ud Wells Wavelen
528. rate Once late Last Fead 17 AM 7716799 B C D E F G H Wavelength Combination Lrm1 Data Mode Absorbance of of 0 5 IJ D 8 o M12 Figure 7 12 Endpoint Data Reduced Displayed as Gray Scale 7 17 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 1 B 18 Graphing Wells You can enlarge the display of the wells shown with Kinetic Spectrum or well scan modes to get a close up view of the data This is done by either double clicking the particular well or cuvette in the data display or by select ing the well or cuvette and then clicking the Well Graph button in the Plate section or CuvetteSet section tool bar Kinetic and Spectrum Well Graphs Well Graphs plot the data for the individual wells in a microplate or for indi vidual cuvettes and show the well ID and reduction information including the goodness of fit R 2 value if appropriate The default display for the X axis shows time for Kinetic plots and wavelength for Spectrum plots OD or 76 T is displayed on the Y axis for both plots Positioning the cursor within a Well Graph shows X and Y values for the cur sor location allowing you to determine more precisely the location of data points Well Graph Options Along the bottom of each Well Graph dialog box are eight buttons five when using well scan mode that perform the following functions e Print Opens the Print dialog box e Reduction Op
529. rator Enterprise Administrator NOA Your MDC Representative will not be able to install SoftMax Pro Enterprise Edition or Enterprise Administrator software since this can be done only by the administrator Pre Installation Information Prior to installing Enterprise Administrator your group or lab must designate an administrator who will install the software and manage the SoftMax Pro users We recommend that this administrator be someone who understands SoftMax Pro well or who is willing to learn SoftMax Pro extensively since the administrator will manage and activate change permissions that control what all of the users can do in SoftMax Pro The administrator can also bea team for example a member from your IT group working together with a person who understands SoftMax Pro Your company may need to create an operating procedure allowing a backup person to act as administrator in case the administrator is unavailable for an extended period of time A KANOS 1f someone tries to log in 3 times using an incorrect User ID Password combination a security feature in SoftMax Pro will invali date the file path linking SoftMax Pro and the Enterprise Administra tor software It will not be possible to work with SoftMax Pro or use it to operate a microplate reader until the administrator logs in cor rectly physically locates the user s PC and resets the path In order to reconnect the administrator must enter the correct user ID and pa
530. rators that are understood by SoftMax Pro A list of these accessors and operators is given at the end of this chapter Resizing and Hiding Columns SoftMax Pro User s Manual The columns in a Group section can be resized in several ways One of the simplest is to drag the line dividing the columns to another position moving it horizontally When you point to a line between columns the cursor changes to a double arrow with a vertical line through it showing that it is ready to move left or right see Figure 8 25 ka Standards Double headed Sample Concentra Aes MeanValue Std Dew CVE arrow cursor Smallest standard value Largest standard value Figure 8 25 Cursor Positioned on a Line between Columns Reducing Column Size and Hiding Columns You can reduce a column to a size smaller than the width of its title in which case the title will not be completely visible You can also hide a column by reducing its size completely drag one edge of the column on top of the other or by selecting that column and choosing Hide from the Group menu If you hide a column in this way and then wish to view it again use the Show All command in the Group menu All columns that were hidden will again be visible There is no way to show only one hidden column Enlarging Column Size You can enlarge the width of columns to any size Increasing the size of col umns may make the width of the Group section increase beyond the page w
531. rbance or Transmit tance Default Protocol A protocol file included with the SoftMax Pro program or created by a user that resides in the default folder The Default Protocol file defines the initial information that will appear when you open SoftMax Pro or when you create a new file from within the program Both Macintosh and Windows versions of this file are called Default Protocol Windows versions append the 3 character ppr extension Desktop The background on which windows and icons appear Dialog Box A rectangular window that appears temporarily to request information Some dialog boxes have options you must choose before SoftMax Pro can carry out a command and must be closed before you can continue in the software Dilution Factor Specifies the amount of dilution for a given sample This factor can be multi plied against interpolated values to determine concentration of undiluted sample Dimmed Unavailable or disabled A dimmed button or command is displayed in light gray instead of black and it cannot be chosen SoftMax Pro User s Manual B 5 Appendix B Glossary of Terms B 6 Double Click To rapidly press and release the mouse button twice while the pointer is on an object Double clicking carries out an action such as opening an icon Drag To move an object on the screen by clicking it holding down the mouse but ton and then moving the mouse Also to select an item in a list by dragging throu
532. rding formulas and the information they can contain can be found in Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using For a complete discussion of writing formulas in SoftMax Pro as well as detailed examples of formulas and customized proto cols refer to the Formula Reference Guide SoftMax Pro User s Manual Chapter 10 Tutorials E Hide Replicates No replicates are used in the FLEX mode tutorial for the EC 50 anal ysis To view and learn about showing and hiding replicates you will be asked to open a different data file If you do not wish to do this please skip this section and go on to Data Analysis Graph on page 10 64 Open the file named Endtut pda located in the Tutorial folder Click in the Standards section to make it active Choose Hide Replicates from the Group menu The display before and after hiding replicates is shown in Figure 10 37 NT ffi Standards Ls E4 fa Standards pg ml Concentratio BackCalcC Well Values MeanValue Std Dev CV 1 362 B1 0 739 0 002 1 368 B2 1 009 C1 0 550 0 003 Standards L017 C2 0 801 D1 0 434 0 003 section 0 794 D2 Showing 0 504 E1 0 276 0 001 0 506 E2 Replicates 0 192 F1 0109 0 001 Default 0195 F2 0 087 G1 0 054 0 004 0 097 G2 0 017 H1 0 004 0 001 17 7 0 015 H2 smallest standard value 0 004 Largest standard value 0 739 NT iii Standards Ls 24 af
533. rds Plots You can add as many plots to a graph as you wish and these can be of the same or different types of groups If you create a graph and later wish to add one or more plots to it simply click the Graph Options button on the Graph section tool bar and then click the New button This will reopen the Plots dialog box allowing you to add or modify the plot information 8 50 SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Error Bars When data is displayed as a scatter or bar graph you can choose to display error bars for the plot of the data for the X and or Y axis Error bars are lines that extend beyond a plotted value in either or both directions and graphi cally represent some amount of error in plotted data v 7 Grapnit3 Fit s E Graph 2 0 93 0 83 0 73 Error Bars 0 63 0 53 0 43 1 Concentration yz amp D I 14 x C B D A Plot 1 Standards1 Concentration vs MeanYalue 0 416 Plot 3 Standards3 Concentration vs Mean alue 0 62 L1 Plot 2 Standards2 Concentration vs Mean alue 0 925 Figure 8 38 Plot Showing Error Bars Choices for displaying error bars relative to a particular axis are the same as those available for displaying data on that axis any column from that group that contains numerical information not columns with text To display error bars click and hold the box next to Error Bars in th
534. re automatically exported as time based data Including labels will affect all data exported but is most noticeable with Group section data since the column headings in the Group table will be shown as will any other calculated data in summaries etc Labels includ ed with Plate and CuvetteSet sections show 1 12 headers for 96 well Plate section data represented as an 8 x 12 matrix 384 well plate data will show 1 24 headers CuvetteSet section labels show A1 A2 etc rep resenting the same number of cuvettes shown in the CuvetteSet section The checkbox for Interpolate Wells is available only for the FlexStation in strument This interpolation is different from the interpolation found in the reduction dialog box see Interpolate Raw Data Checkbox on page 8 34 This checkbox is used to normalize the kinetic read times of other wells against the read time of the first well in each column Because injec tion occurs at the same time in all wells of a column but the wells are read sequentially the reaction advances in the other wells while the first is be ing read Consequently the data in the other wells must be interpolated against the amount of time that has elapsed between injection and reading in order to fairly compare data from one well to another and also to com pare data from one run to another Note that well interpolated data cannot be reimported into SoftMax Pro Add Filename and Date will include this informa
535. re serial number can be found on the original SoftMax Pro CD on the inside cover of this manual and on the soft ware Warranty Registration Card please remember to fill out this card and return it to Molecular Devices Corporation The instrument serial number S N is located on a label affixed to the back of the instrument Type the information in the spaces provided and then click OK Note that the option to click will not be available until all information has been entered Molecular Devices Please register this copy of S FTImazs Pro by providing the following information Software Serial Number e Instrument Serial Number S Nk OK Figure 2 4 Registration Dialog Box After all information has been entered correctly and you click OK a title screen will appear see Figure 2 5 showing the registration information you entered If you have difficulty entering the registration information refer to Chapter 11 Troubleshooting for assistance 2 13 Chapter 2 Installation 2 14 Py O O O SOFTmax PRO Drug Discovery Edition ry SOFTmax PRO Life Sciences Edition Figure 2 5 SoftMax Pro Title Screen Examples After a few seconds the title screen will be replaced by an untitled SoftMax Pro window see Figure 2 6 The screen you see may be different depending upon the type of instrument you are using Pa Untitled lolx WF L5 Introduction aj E v E notes 24 a Default
536. read plate blanking is chosen SoftMax Pro will prompt you to first read the blank plate after you click the Read button Automix amp Blanking pos Atomi MM Before First Read 5 secs The pre read values will be stored in the data file and the individual well values collected during the pre reading of the plate will be subtracted from the individual well values obtained during the actual reading of samples This is particularly useful for readings in the UV Pre read blanking may be turned off in the Reduction dialog box click on the Reduction button so the data may be seen with and without pre read blanking subtracted This option is only available within the Reduction dialog box if pre read blanking was chosen in the instrument setup The purpose of pre read plate blanks is to correct for well to well variability and in the case of PathCheck to subtract plate background before applying pathlength correction A background constant subtraction setting is also available for instruments capable of using PathCheck in the Instrument Settings gt PathCheck option If you pre read a plate you should not use a background constant subtraction and vice versa Pre read blanking is available for all read modes but is most useful for Endpoint readings When used in Spectrum scan mode SpectraMax instru ments only the pre read scan is subtracted on a point by point basis from the normal scan If pre read is sele
537. ready chosen and then close the dialog box A synopsis of the steps you will take during this tutorial is given below Create a new file Instrument setup Template setup Set reduction parameters Set display parameters Save the protocol Read the plate start the simulator and then open the Tutorial file Analyze data Standard Curve Analyze data Standard section Print the report re eer sp scar ps 9 T9 When you install SoftMax Pro you are asked to choose between absorbance fluorescence or FlexStation installation The choice made at that time will deter mine the type of default protocol file that is installed Tutorial 1 pertains to users of either absorbance or fluorescence instruments Tutorial 2 later in this chapter deals with the FlexStation instrument 10 4 SoftMax Pro User s Manual Chapter 10 Tutorials Description of the Assay Example Following are the details about the quantitative assay settings we will use to create a protocol file using SoftMax Pro Read Mode Standards in ug ml Standard Curve Fit Blanks Auto Mix Autocalibrate Endpoint 0 0 1 0 25 0 5 0 75 1 0 and 1 4 each standard run in duplicate Linear Plate blank run in duplicate On On Instrument Settings Specific to Absorbance Instruments Wavelengths otrips One 405 nm Read entire plate Instrument Settings Specific to Fluorescence Instruments Wavelengths Sensitivity A
538. reduction parameters or display parameters as you wish or if you didn t set them up in Step 4 do so now Save the data file Analyze the data using the Group section tables and Graph sections Save the data file when you are finished This chapter discusses how to define instrument settings create a template and initiate a reading Data analysis is covered in Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 4 3 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 1 k Instrument Settings Not all instrument settings are available with all instruments Only the settings that pertain to the instrument that is connected to the computer or the one chosen in the Preferences will be shown in the Instrument Settings dialog box You must specify the instrument settings prior to actually reading a micro plate or cuvette These settings include Microplate or Cuvette e The read mode Endpoint Kinetic or Spectrum e Wavelength s used with Endpoint and Kinetic mode or the wavelength range for Spectrum mode e Timing for Kinetic runs e Speed Read SpectraMax Plus Plus 94 190 and 340PC only Microplate Only e Automix all instruments except Emax e Blanking pre read plate e Pathlength correction using the SpectraMax Plus Plus 94 190 and 340PC only e Strips SpectraMax and VersaMax instruments only e Autocalibrate Settings for the reading are made in the Instrument Sett
539. rently depending upon whether or not you are setting a password for the first time or if you are changing removing a pass word If you have not previously set a password the dialog box will appear like that on the left of Figure 9 17 To set a password type it in both boxes first to set it and then to confirm it After an Author s name has been entered this name will appear at the lower left of the Plate or CuvetteSet sec tion When a file has been password protected an icon showing a lock will also appear at the top right of the title bar of the SoftMax Pro program gj When changing or removing a password the dialog box will appear as shown on the right of Figure 9 17 Before you can change or remove the password you are required to enter the old password in the top text box After entering the old password checking the box next to Remove Password and typing nothing in the rest of the boxes will clear the password from the file when you click OK Otherwise entering a new password and confirming it with a second entry will update the password protection Be sure to choose a password that you will be able to remember since the file will be locked to changes of nearly any kind should you forget it If you try to perform an unauthorized action on a password protected file a dialog box will appear asking you to enter the password OXON SoftMax Pro provides no means of overriding password protection If you are concerned that yo
540. rescence only and the choice for bottom read with the FlexStation instrument only e Assay plate type number of wells e Automix e Wells to read e Autocalibration Settings for the reading are made in the Instrument Settings dialog box which can be accessed in three ways 1 Choose Instrument Setup from the Control menu 2 Click the Setup button in the tool bar of the active expanded Plate section or 3 Double click anywhere in the settings box located to the right of the well grid in the active expanded Plate section You may create more than one Plate section in a single experiment and the instrument settings associated with each Plate section can be different Instrument Setup can only be chosen while a Plate section is active and the settings you choose will affect only that section A new Plate section that is created in an experiment that contains an active Plate section will contain the same instrument settings found in the previously active section The settings for the new section can be modified as desired 5 4 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Table 5 1 lists all possible instrument settings choices based on instrument type When you have defined the instrument settings click the button to close the Instrument Settings dialog box After closing a summary of the instrument settings will appear in the box to the right of the data display in th
541. ressed button raw values without a reduced number SoftMax Pro User s Manual 10 23 Chapter 10 Tutorials Click the button The following options will appear L Display Grayscale k cma oook Figure 10 25 Display Options for Reduced Displays Absorbance shown Choose Grayscale from the drop down menu that appears when you click the Dis play options menu The dialog box will update to show the low and high limits for grayscale display values which are automatically set to the minimum and maximum data values mw Display L1 lani bowand ues Limit Settings Figure 10 26 Display Dialog Box Updated with Low and High Limit Settings Absorbance shown 10 24 SoftMax Pro User s Manual Chapter 10 Tutorials Click OK The dialog box will close and the data display in the Plate section will update to show the data represented by shades of gray as shown in Fig ure 10 27 Je ses 1102 iE Plate 1 S555 EJ 5S roc Automix Off Calibrate On a 12 11 PM 2 4 00 G l NE Ji E NM Pre Read Used Mo Data JE aan Wavelength Combination Lrm1 Data Made Absorbance Jeon feo2 feos neos as B o5 Emo Re o Figure 10 27 Plate Display with Data Displayed as Gray Scale Absorbance shown Step 8 Data Analysis Group Sections SoftMax Pro User s Manual Group sections are created automatically by SoftMax Pro when you define gr
542. rived from 7 64 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments that transformed data Because of this you should be cautious about using correlation coefficients as an indicator of goodness of fit Do not let this single value for goodness of fit sway your intuition about which fit is best Some curve fits may seemingly give a good fit e g fit values close to 1 but when inspected by eye show a poor fit along a major portion of the plot Graph Options Graphs can be customized further through the Graph Options dialog box which is accessed by choosing Graph Type from the Graph menu or by click ing the Graph button Jin the section tool bar Graph Options Title Graph Height Type SU Scatter Sample Text m Mh 3 Cluster bar Sample Text Set hl Stack bar Legend no Connect points 5 le Text nen Sets Flot symbols D Flot O Plot 2 MaluesmeStandards A Errorz Wo Error Y Errors Mo Error Figure 7 52 Graph Options Dialog Box The default options are shown in Figure 7 52 but you can alter these as desired you may change the font and type size used for the title legend and or axes of the graph and you can choose the color and type of symbol used on a scatter graph to show the plot The graphs shown in preceding Fig ures reflect the default graph options Connecting the points of a scatter graph can be useful in determining the closeness of
543. roups on a graph even if the groups are in sepa rate experiments It is possible for example to plot multiple dilution series of an unknown to graph together a series of patient samples or to plot several controls that have been run over time Following is asummary of the steps required to create multiple plots within a single graph after which is an example that provides more detail Multiple Plots Step Summary Create all required groups in the Template Editor e Read the microplate or cuvette to obtain data e Create a graph section e Add plots to the graph and choose the group and the X and Y values to be shown for each plot Example of Creating Multiple Plots This example shows how three groups all of which are Standards were plot ted on the same graph 1 Groups are created in the Template Editor For this example four groups are cre ated three for standards Standard1 Standard2 and Standard3 and one for Unknowns Unknownl SoftMax Pro User s Manual 7 49 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Experiments 1 Plate 1 Broun ais Sample Unko1 Jw Series f Assign Clear 10 11 12 Cancer 9k Figure 7 36 Standards and Unknowns Groups Created 2 The associated Group sections are created using the Standards column format which by default generates columns for concentration and optical density val ues The example below shows t
544. rs If the sample name is entirely numeric it will not be truncated If sample names are very long and they must be kept long for reference you will have to set up your replicates manually and will not be able to use the series function 4 32 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Blanking In addition to the pre read plate blanking selection found in the Instrument Settings dialog box four more types of blanking are available in SoftMax Pro Understanding the differences between the types of blanking functions will enable you to choose the type of blanking that is appropriate for your assay Note that blanks can be used in combination and have cumulative effects The five types of blanking that are defined in SoftMax Pro are e Pre read plate blank Plate section discussed earlier on page 4 19 e Ref CuvetteSet section e Plate blank Plate section Template blank CuvetteSet section e Group blank Plate section and CuvetteSet section Ref CuvetteSet section only Performing a reference reading of the cuvette causes the SpectraMax Plus or Plus to take a reading of the cuvette in the cuvette port A single reference reading can be used with one or more cuvettes in a CuvetteSet section the default applies to all cuvettes in the set or different reference readings can be taken and applied to different cuvettes in the set This reference read is then app
545. rs appear in the name labels above individual cuvettes when dis played as one or three cuvettes per row or when colored areas appear when the CuvetteSet section is displayed as a grid this means a template has been defined for the CuvetteSet section Each group defined in the template has a different color the icon of the corresponding group table will have the same color If your experiment requires multiple cuvettes you can add up to 96 cuvettes in a single CuvetteSet section and or create more than one CuvetteSet sec tion The CuvetteSet menu contains the items shown in Table 3 3 shown with their tool bar or mouse equivalents These menu items and buttons are active only when a CuvetteSet section is active Chapter 3 SoftMax Pro Overview l F Table 3 3 CuvetteSet Section Menu Commands and Tool Bar Buttons Menu Command Description Tool Bar Mouse Equivalent P Button avem cud Ie Opens the CuvetteSet section in a new window double click lt 5 EMPM CuvetteSetti ame of Cuvetteset section pens the section dialog box dowblescliel Instrument Setup in the Control Opens the Instrument Settings dialog box for menu this CuvetteSet section Setup Opens the Template dialog box allowing you Template in the CuvetteSet menu EE to create or edit the template ES Template Opens the Reduction dialog box containing Reduction in the CuvetteSet menu E onas Enlarges selected well s to a Well Graph
546. rst file saved on January 31 2001 would have the name Data 01 31 2001 1 Note that if a time is not appended see the next item SoftMax Pro indexes the runs for that day starting with 1 and incrementing upward with each successive run Append Time Enabling the checkbox for Append Time will include the time in the auto matic file name for the saved data file in the format hhmmss For example using the file prefix Data the file saved at 10 12 38 a m would have the name Data 101238 Autoprint Checking this box will enable automatic printing of a report after each plate is read This report will contain information from each section that is designated to be included in the report the default is15 for all sections to be included For more information on reports and including or excluding sections see Chapter 3 SoftMax Pro Overview 2 22 SoftMax Pro User s Manual Chapter 2 Installation Assays Folder SoftMax Pro User s Manual The Assays menu is used to choose the folder that will contain protocol files you wish to access most often After installation of SoftMax Pro the protocol files shown under the Assays menu will be the basic protocols shipped with the software Other folders are installed with SoftMax Pro containing protocol files for other types assays you may select one of these folders instead if desired To change the default folder choose Assays and then choose Set Folder
547. rt Export 9 21 Importing data 9 21 Incubator command 3 31 Incubator button 3 8 Installation 2 8 Installation Enterprise Administrator 6 7 Instrument settings 3 23 4 4 5 4 5 38 10 9 10 38 mode 4 9 5 10 5 40 read type 5 13 summary 4 5 5 5 wavelengths 4 11 Instrument Setup command 3 31 Interpolate Raw Data 8 34 Interpolate Wells 9 16 K Kinetic 1 3 4 10 5 11 7 28 8 21 display defaults 7 6 8 6 interval 4 10 5 11 5 44 limits 7 28 8 21 negative OD values 7 28 8 21 reduction 7 28 7 33 8 21 8 26 8 30 run time 4 10 5 11 5 44 L Lag Time 7 28 7 30 8 21 8 23 Lambda at Maximum 7 37 8 30 Lambda at Maximum Minimum 7 37 8 30 Large Display B 8 License Enterprise Administrator 6 26 License Page 6 13 SoftMax Pro User s Manual Limits data display 7 15 8 11 MaxOD 7 28 7 36 8 21 8 29 MinOD 7 36 8 29 Linear curve fit 7 55 8 53 B 8 Lock software after three logon attempts 6 10 Log Log curve fit 7 57 8 55 Log Logit curve fit 7 59 8 57 B 9 Luminescence B 9 M Macintosh computer 2 6 memory insufficient 11 16 problems 11 15 software 2 8 Mask 7 22 8 18 button 7 22 8 18 command 3 33 3 35 7 22 8 18 wells or cuvettes 7 22 8 18 Mask command 7 22 8 18 Max Lambda 7 37 8 30 Maximum 7 36 8 29 Maximum reduction B 9 Max Min 8 33 MaxOD 7 28 7 32 7 36 8 21 8 29 B 9 MaxRFU RLU 8 21 8 25 B 9 Mean 8 33 Memory insufficient Macintosh 11 16 Menu CuvetteSet 3 35 Experi
548. rval SoftMax Pro User s Manual Data is collected over time with multiple readings taken at regular intervals The default reduced values calculated for Kinetic data are Vmax per Sec RFU Sec or RLU Sec Time to Vmax seconds Onset Time seconds Time at Minimum Time at Maximum Time at 1 2 Maximum Slope and Area Under Curve E Instrument Settings Oi HM Endpoint Kinetic Spectrum Options Read Type Fluorescence Run Time 2 Humber af Reads 34 405 450 Sensitivity Readings 6 PMT Medium Minimum Interval 0 36 Time 5 00 Interval 0 09 Reads 24 Minimum Interval 0 36 Automix Before Off Between Off AutoCa librate On na Kinetic interval tao short Plate Type Figure 5 6 Instrument Settings for a Kinetic Reading Timing When Kinetic mode is chosen the Timing area becomes active allowing you to enter the total Kinetic run time and the time interval between readings A default value will be shown for the run time to change this value click in the box Up and down arrows will appear enabling you to increase or decrease the value shown Highlight the hours minutes or seconds shown and then click the up or down arrow until the desired value is reached Alter natively you can type over an existing value using the keyboard After choosing a total run time and an interval between readings the total number of readings that will result is shown below the run time If you set the interval
549. s TO aM ocu 6 Well Costar AutoRead Turn autoread on Delay n secs Figure 5 4 Instrument Settings Dialog Boxes for Well Scan Mode SoftMax Pro User s Manual 5 9 Chapter 5 Reading Using Fluorescence or Luminescence Instruments k Instrument Settings Options Read Mode Choices for the read mode are Endpoint Kinetic Spectrum or Well Scan To select a read mode click the appropriate section in the Instrument Settings dialog box Endpoint A single reading of the microplate can be taken at a single or multiple wave length s Values reported are relative fluorescence units RFU or relative luminescence units RLU The default reduced value is Lm1 or Lm2 Lm4 if multiple wavelengths were chosen m Instrument Settings E Endpoint Kinetic Spectrum Well Scan Read Mode Buttons Options Fluorescence Wavelengths Ex Em Auto Cutoff On Fluorescence RFUs Vus ode e Luminescence RLUs Sensitivity Readings 6 i C Time Resolved RFUs PMT Auto Autornix Before Off AutoCalibrate On Assay Plate Type 96 Vell Standard Wells To Read Read entire plate Figure 5 5 Instrument Settings for an Endpoint Reading You can choose up to four wavelengths 5 10 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Kinetic Number of Readings Calculated from the Run Time and Inte
550. s 4 24 fe Standards pg ml Concentrat MeanValue Std Dev CV After Hiding Replicates Smallest standard value 0 004 Largest standard value 0 739 Figure 10 37 Standard Section Before and After Hide Replicates More information about modifying group sections can be found in Chapters 7 or 8 choose the chapter that is appropriate for the instrument you are using Step 9 Data Analysis Standard Curve The Default Protocol used to create this tutorial example file contains the pre defined groups Standards Samples and Controls during the course of this tutorial you also created a new group called Sam w dil with dilution In addition it contains a Graph section which has been created to display a stan dard curve Scroll down within the SoftMax Pro window until you see the section tool bar for Graph 1 This section is closed click the indicator on the left side of the tool bar to open it This graph contains a single plot as shown in Figure 10 38 SoftMax Pro User s Manual 10 31 Chapter 10 Tutorials v 2 Graph ji Graph 1 Mean alue 0 0 2 o Std Standards Concentration vs bWeanwaluej 0 4 0 6 0 5 1 1 2 1 4 Concentration Figure 10 38 Standard Curve Graph Section No Fit Applied This graph is set to show concentration on the X axis and mean value on the Y axis If you are continuing with simulated data no fit will have been applied the Endtut pdf file has a linear fit ap
551. s Well 6 Plate Last Read Ymax units per sec Plate 1 Kinetic Time Interval 5 00 0 37 Reads 9 Fluorescence Ex Em Cutoff Lm 405 450 435 x Auto Cutoff Automix Off Ir OO m m oO 0 UJ Wavelength Combination Lm1 v Plate k r4 Calibrate Once PMT Medium Spectrum Display Lambda at Maximum Plate 1 Spectrum Fluorescence Excitation Cutoff EmStart Stop Step 350 None 350 750 10 Automix Off Calibrate Once NT Plate 1 Em PMT Auto Reads Well 6 Average 6 Plate 1 rd well Scan Pattern Fill Density 3 Spacing 1 13mm Total Points 9 Fluorescence Ex Em Cutoff Well Scan Lm1 485 538 530 T Om moo m r Display FLEX 8 10 NT Plates Auto Cutoff Automix Off Eis i A Vmax units per sec Plate 1 Aquarius 5 00 0 09 Time Interval Reads 34 Fluorescence Bottom read Ex Em Cutoff Lm1 485 538 530 SNINISINIKKK SSIISSKNI Auto Cutoff Wavelength Combination Lm1 Trans1 H 100p R21 v 21001 6010 Automix Off Calibrate On PMT Medium
552. s must change their passwords When a user logs on within 15 days of pass word expiration notification of the need to change the password will appear If the password is not changed before the expiration date it will expire and the user will be unable to log on The administrator must then reset the pass word Idle Time out indicates the duration of inactive time in minutes after which the software application such as SoftMax Pro will automatically log out Lock software after three logon attempts When this option is enabled if a user enters the wrong password three times in succession the software will lock A message will appear explaining that the program is locked and that the administrator must enable the path to the database again Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator Enterprise Edition of SoftMax Pro Security Menu The Security menu provides the initial connection from SoftMax Pro Enter prise Edition to the Enterprise Administrator database Security Set Administrative Group Log Out Change Password Show Audit Trail Show Admin Info Figure 6 8 Security Menu in SoftMax Pro Enterprise Edition Set Administrative Group Displays the Open dialog box used to select the Enterprise Administrator database file that contains the user and password information for SoftMax Pro Enterprise Edition Log On Prompts a user to enter his her assigned user name and
553. s without additional warranties of any kind either express or implied including but not limited to any implied warranty of merchantability or fitness for a particular purpose The entire risk as to the quality and performance of the Software is the User s MDC does not warrant that the functions contained in the Software will meet the User s requirements nor that the operation of the pro gram will be uninterrupted or error free Some states do not allow the exclusion of implied warranties and therefore the above exclusion may not apply to you LIMITATION OF REMEDIES MDC s entire liability and the User s only remedy with respect to the Limited Warranty set forth above is 1 The replacement of any discs not meeting the Limited Warranty set forth above provided such discs are returned to MDC with evidence of date of purchase or 2 If MDC is unable to deliver a replacement disc which is free of defects in materials and workmanship the user may terminate this license agreement upon return of all materials including manual and disc s to MDC for a full refund of the purchase price In no event will MDC be liable to the User for any damages including any lost profits lost savings or other incidental or consequential damages arising out of the use or inability to use the software even if MDC has been advised of the possibility of such damages or for any claim by another party Some states do not allow the limitation or exclusion of liability for
554. s Manual 13 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 14 NT E CuvetteSet 1 epi le CuvetteSet 1 A1 1 A S a 5 Endpoint 0 000 OD 0 000 OD 0 000 OD Display Data 7 16 39 8 58 AM Data 7 16 39 8 57 AI Data 7 16 39 8 57 AM Ref 716 99 8 57 AM Ref 7 16 9292 8 57 AM Ref 7 16 92 8 57 AM Wavelength Combination Lra Data Mode Absorbance ve EE cuvettesetsti Ymax CuvetteSet 1 DY a Rin tic Time 010 1 1 Interval 0 20 agate ep pa m Reads 16 Disp ay Data 7 15 29 9 02 4M Data 7 16 99 9 02 AP Data 7 16 99 9 02 AM Lmi 450 Ref 716 99 3 02 AP Ref 7 16 99 3 02 4M Ref 7 16 99 9 07 AM Lag Time 0 00 Wavelength Combination ILm1 stint zog Data Mode Absorbance DD Max 1 Vmax Pts 16 16 Lambda at Maximum CuvetteSet 1 mY J Spectrum Start 450 pue AAI U AAI SS Display Step 15 Data 7716799 9 04 AM Data 7 16 99 3 05 AM Data 7 16 99 9 05 AH Ref No Reference Ref Mo Reference Ref Mo Reference Start 450nm End S nm Wavelength Combination Lm1 T alee Data Mode Absorbance i Figure 7 8 Default Displays for a CuvetteSet Section Endpoint Kinetic and Spectrum Modes Reduced Number The reduced number is based on the settings defined in the Reduction dialog box It is a combination of blank subtraction pre read and or plate blank in the Plate section and ref and or template blank in the CuvetteSet section wavelength reduction and
555. s Vmax Rate if you have modified the Vmax Points Area Under Curve This reduction estimates the area under the curve as defined by the data plots in wells within the reduction limits The data plots are treated as a series of trapezoids with vertices at successive data points and at the X axis coordi nates of the data points The areas defined by each of the trapezoids are then computed and summed Mean This setting takes the average of all data points and returns that value Smoothing irm Smoothing SITO NT OEDEEEPOELEEETEECEEHTEDEHEEOLEDLELCELO RET OHEEEHTUERECOE EDENDI HEECOCOEEEG Moving Average x Points Figure 8 19 FLEX Mode Reduction Dialog Box Smoothing Setting Smoothing is used to reduce noise in FLEX readings This setting takes the moving average of the raw data and displays the FLEX plot using these aver aged values The default is no smoothing setting of 1 averaged point A setting of 3 provides the least amount of smoothing the value of the center point is added to the values of the points on either side the sum of these points is then divided by three and the resulting value is substituted for the center point A setting of 9 would average the center point with the four points on each side When multiple wavelengths are read it is important to note that smoothing is performed before performing wavelength combination calculations such as ratios or multiplication For example if you read
556. s each time a new plot is added to a Graph section In this dialog box you can rename the plot assign specific information to be plotted on the X and Y axes and choose which icon and color will be used If you rename the plot the title of the dialog box will change to show the new plot name All groups that have been created or assigned within the file are listed on the left side of the Source field in the Plot X dialog box The first group in the file is automatically selected Other groups can be selected by highlighting them When a group is selected the pull down menus in the X axis and Y axis subfields change to show the group columns that are available for graphing Group columns can be assigned for both the values to be plotted and any error bars to be included for both axes Summaries and user defined formulas can also be graphed Pushing the zi button for either the X axis or Y axis opens a dialog box which allows you to enter formulas To plot a summary enter the summary name in this dialog box Formulas can be plotted for comparison with other data For example if the data is assumed to follow a specific model the model formula can be entered as a plot and then the data can be chosen for another plot within the same graph You can also use this feature to plot Kinetic or Spectrum data in 7 47 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 1 B 48 Graph sections For example to
557. s in the destination experiment the group Unknown will be created but all information regarding the group Standards in the destination experiment will remain un changed If you copy and paste the template with the same experi ment no new groups are created Group names can be edited in the pasted template these changes will be reflected in the original tem plate also Export Template Opens the Export Template to dialog allowing you to ex port a template to a tab delimited ASCII text file Import Template Opens the Import Template to dialog allowing you to import a template from a tab delimited ASCII text file Section Name Allows you to change the name of the Plate section Mouse Equivalent double click the name of the Plate Plate 1 Jin the Plate section tool bar to the right of the Plate section icon New Window Opens the active section in a separate window Mouse Equivalent double click the icon in the Plate section tool bar SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview CuvetteSet Menu New Cuvette Template Reduction Display braph Copy Template Paste Template Expart Template Import Template Section Name New Window SoftMax Pro User s Manual This menu item changes to show CuvetteSet Plate Notes Graph or Group depending on the type of section that is currently active If no section is active this me
558. s of the excited light to overlap the emission Spectrum allowing better selection and quantitation of the emitted light See the instru ment manual for the SpectraMax Gemini fluorescence reader or FlexStation instrument for more information regarding cutoff filter settings Sensitivity Readings This setting determines the number of readings that will be performed on each well of the microplate readings are averaged and the average reading is displayed rU Readings aaa rU Readings S E SKEEREENENUNRAUA NEUEN UE Fast Normal Precise Fast Normal Precise Ce DS RERRRRRRERERRERERERERRERERRRRARAEERARAEMAEE Figure 5 12 Number of Readings Setting The left shows the default setting for fluorescence the right shows changing this set ting for more precision with a greater number of readings per well The default number of readings per well varies with the read type for fluores cence the default is 6 for luminescence the default is 30 for time resolved flu orescence the default is 20 The number of readings is variable from 1 to 30 With the luminescence read mode the integration time is directly proportional to the number of readings per well determined by this setting Each reading per well is equal to 3 2 milliseconds of integra tion time PMT Sensitivity The PMT photomultiplier tube is a vacuum tube that detects light espe cially from dim sources through the use of photoemission and successive instances of secon
559. s or Cuvettes in the Template Editor In the Template Editor for a Plate section or a CuvetteSet section you can select one or more wells or cuvettes by clicking or dragging on the grid Mul tiple contiguous wells or cuvettes can be selected by dragging across them Non contiguous selections can be made by holding down the Shift key and clicking individual wells or cuvettes Clicking a row label will select the entire row clicking a column label will select the entire column Contiguous col umns or rows 1 12 or A H for a 96 well plate for example can also be selected by dragging Non contiguous columns or rows can be selected by holding the 3 command key on the Macintosh or the CTRL key in Windows and clicking in labels Double clicking anywhere within a group will select the entire group To deselect all wells quickly click in the empty border around the Template e When you leave the Template Editor by clicking the button the Plate CuvetteSet section will show the boundaries of the groups created in the template Group labels and sample descriptors will not be visible you can see them by holding down the Ctrl Shift keys When you release the key s the original view of the Plate section or CuvetteSet section will return SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments The Template Editor Tool Bar The tool bar will appear differently depending upon what type of
560. s set in Enterprise Administrator determine the options available to users of SoftMax Pro Enterprise Edition e Overwrite existing files e Add E signatures e Add modify E statements e Clear audit trail e Change Autosave parameters e Change Group table and template parameters e Change reduction settings e Mask unmask wells e Read once e Delete data e Remove E signatures More information about these options can be found on page 6 12 6 3 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator The administrator does not have access to user passwords Pass words require a minimum of 6 characters and an alphabetic and numeric combination The administrator may change a user s pass word however 6 4 SoftMax Pro User s Manual Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administrator SoftMax Pro Permission in Enterprise Administrator Save files and protocols Change Instrument Settings oign e statements Change Notes Text Create and modify e statements Mask UnMask Wells Clear Audit Trail Change Auto save parameters Read Empty Plate and Cuvette Sections Overwrite Plate and Cuvette Data Change Group Table and Template settings Remove signatures from e statements C Hu rnm c Ww Change Data Reduction Settings es A M User Privile ADIIT m J S gt 3 Y z Q 2 Q x uo 3 a T O1 Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Administ
561. s that customers consult a third party automation company if internal software resources or expertise are not available All leading automation vendors have integrated SoftMax Pro and Molecular Devices instruments with their systems A partial list of these vendors includes e Beckman Coulter SAGIAN e Zymark Corporation e CRS Robotics Corporation e TekCel Corporation e OIAGEN Inc e Hudson Control Group If you are a robotic vendor or an individual integrator and you need more information please contact your local Molecular Devices sales representative or send an email with the message titled SoftMax Pro describing your interest to info moldev com SoftMax Pro User s Manual Chapter 12 SoftMax Pro Remote Command Language SoftMax Pro Commands SoftMax Pro commands are single words If parameters are required with a command a colon is used between the command and the parameters Commands may be sent using either the WM SETTEXT or WM COPYDATA approach Completion messages or data are returned directly when com mands are sent using the WM COPYDATA method Completion messages or data are placed in the system clipboard when commands are sent using the WM SETTEXT method ClearQueue Close When queued processing is enabled this command clears the queue of remote commands that have not yet been executed This command can be useful when you wish to clear the queue perhaps during software develop ment If queued
562. s way you can use the same template for more than one CuvetteSet section or you can modify the pasted tem plate as desired If you are pasting a template into a new experiment and groups from the source template do not exist in the destination experiment pasting the template will create them Existing groups of the same name will not be changed by the pasting process For example if the template be ing copied contains the groups Standards and Unknown and the group Standards already exists in the destination experiment the group Unknown will be created but all information regarding the group Standards in the destination experiment will remain un changed If you copy and paste the template with the same experi ment no new groups are created Group names can be edited in the pasted template these changes will be reflected in the original tem plate also Export Template Opens the Export Template to dialog allowing you to ex port a template to a tab delimited ASCII text file Import Template Opens the Import Template to dialog allowing you to import a template from a tab delimited ASCII text file Section Name Allows you to change the name of the CuvetteSet section Mouse Equivalent double click the name of the CuvetteSet section CuvetteSet 1 to the right of the CuvetteSet section icon New Window Opens the active section in a separate window Mouse Equivalent double click the f T icon in the Cuve
563. sage hwnd WM COPYDATA tag LPARAM amp cd Either a completion message or return data is returned Remote commands Return Status Return Timing and ReturnData return data In either case data is received through an asynchronous windows message inside Win32 COPY DATASTRUCT type data packet For example a typical OnCopyData window callback is shown below where the string data retrieved is finally stored into a Microsoft CString object Note the use of variable replyTag discussed above which is used to isolate the correct windows message returned BOOL CUserDlg OnCopyData CWnd pWnd COPYDATASTRUCT cd if cd gt dwData replyTag String pointing to status CString retStatus char cd gt lpData i SoftMax Pro User s Manual Chapter 12 SoftMax Pro Remote Command Language sent Summarizing Remote Commanding SoftMax Pro has two groups of remote command instructions 1 Remote commands that DO NOT return data Commands that do not receive data are further broken into A The user wishes to receive a completion message after the command has been After receiving a remote command that uused the WM_COPYDATA mes sage SoftMax Pro will send a completion message once the command has been executed An asynchronous window message is returned as a win dow callback typically an OnCopyData handler inside the Win32 COFY DATASTRUCT types data packet For example if the Read command were s
564. se and Urea Bromocreosol ppr Kinetic ELISA K ELISA with AP and pNPP ppr K ELISA with HRP and ABTS ppr K ELISA with HRP and OPD ppr K ELISA with HRP and TMB ppr LAL Kinetic Analyst Qualification ppr Chromogenic Interference Screening ppr Interference Validation ppr Reagent Qualification ppr Routine Testing complete ppr Routine Testing simple ppr RSE CSE Standardization ppr LAL Kinetic Analyst Qualification ppr Turbidimetric Interference Screening ppr Interference validation ppr Reagent Qualification ppr Routine Testing complete ppr Routine Testing simple ppr RSE CSE standardization ppr Nucleic Acids DNA Background Constant ppr DNA Plate Blank Method ppr DNA Pre Read Method ppr Oligreen ppr PicoGreen ppr Antisera Titer ppr Gemini Optimization ppr ID 50 Determination ppr Kinetic With PathCheck ppr Michaelis Menten ppr Multiple Peak Protocol ppr Multiple Peak wavelength and OD ppr Parallel Line Analysis ppr Pipettor Regular Test Method ppr Sample Associated Blank ppr tTest ppr SoftMax Pro User s Manual 2 11 Chapter 2 Installation Table 2 1 Files that Are Installed Protein Assays A280 with cuvette ref ppr A280 with water constant ppr BCA ppr Bradford ppr Lowry ppr NanoOrange ppr Tutorial L Drug Discovery Data Files not included with Life Sciences version L Example Text Files for Analyst System Import Read Me First txt SampleFPData txt SampleFPIAData txt SampleGFactorFPData txt Sample
565. se column formats manually but if you choose to do this the information will not appear automatically in the Group section To see it you will need to create a new column containing the formula ISampledescriptor For more information about creating columns see Adding Columns on page 8 42 Column Format defines the default column types that will be created for a new group or shows the original column format setting that was used to cre ate the group you are editing Four selections are offered Basic Standard Unknowns and Unknowns Dilution Different types of columns will be created for a new group or changed in an existing group depending on which of these column format settings you select Table 5 2 shows the default columns created with each selection type 5 23 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Cin NOJN If you are creating a new group for unknowns that have a dilution factor use the Unknowns dilution column format as a base rather than the Unknowns group included in the default protocol Table 5 2 Columns Created and Their Formulas Formula SampleNames SampleNames ISampleNames ISampleNames a a Formula WellIDs WellIDs WellIDs WellIDs Name Sample Concentration Values Formula Concentration WellValues WellValues Name Values Values R Outside Standard R Outside Standard Range Range Formula WellValues WellValues If Values gt MinStd If Values gt
566. section or CuvetteSet section are performed in the order shown below If an option has either not been selected in the Instrument Settings dialog box is not available for the instrument you are using or has not been defined in the template and or Reduction dialog box SoftMax Pro starts continues with the next listed calculation Plate Section 1 2 3 4 5 6 7 Convert OD to T and vice versa Pre read plate blank or plate background constant subtraction or division for 76T PathCheck pathlength correction different equations for OD and T available with SpectraMax Plus Plus 94 190 and 340PC only Plate blank subtraction Wavelength reduction Kinetic or Spectrum reduction Group blank subtraction CuvetteSet Section 1 2 3 4 5 6 Convert OD to T and vice versa Reference is subtracted from the reading optical density mode or the read data is divided by the reference 76 T mode see Caution below Template blank subtraction Wavelength reduction Kinetic or Spectrum reduction Group blank subtraction SoftMax Pro User s Manual 7 23 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 1 B 24 NISNEGN Separate mathematical calculations are used for handling OD and T calculations for Pre read plate blanking PathCheck and Reference because OD calculations are performed on a linear scale whereas T calculations are performed on a logarithmic scale Other ca
567. see a reduced num ber as well as another type of display click the box next to the option that says With reduced number in the lower left of the dialog box to enable it Chang ing the reduction parameters after choosing a reduced data display mode will cause the display to update accordingly More information about reduction formulas and how to customize them follows later in this chapter SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments TU JA Raw data in 1000 s Plate 1 Normal Display 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 v Gi Piate a Raw data in 1000 s Plate 1 1 2 3 6 7 2 755 3 055 Endpoint 3 355 2 655 Fluorescence 3 955 4 355 Ex Em Cutoff Lmi 355 612 610 Auto Cutoff 4 755 5 255 5 755 6 355 Automix Off 6 955 7 655 Calibrate On PMT Auto Doz Zr RE roa moot 8 455 9 355 Reads well 6 Endpoint Lmi Automix Off Last Read 10 253 1 595 Ex 355 Calibrate On 7 14 99 5 755 6 355 6 955 7 655 8 455 9 355 10 255 11 355 12 455 13 75515 155 Plate Last Read Em 612 PMT Auto 7 99 AM 7 59 AM 7 14 99 Cutoff 610 6 955 7 655 8 455 9 355 110 255 11 355 12 455 13 755 15 155 16 755 18 455 ua Reads Well 6 I ik Auto Cu
568. set ibo dubito debdict oh dh dd Paci adi edd ee qeu d dad 3 25 Instrument Settings Dialog Boxes for an Endpoint Reading 4 6 Instrument Settings Dialog Boxes for a Kinetic Reading 4 7 Instrument Settings Dialog Boxes for a Spectrum Reading 4 8 Instrument Settings for an Endpoint Reading llsleeeel essen 4 9 Instrument Settings for a Kinetic Reading sleleeleeeel esses 4 10 Instrument Settings for a Spectrum Reading esles esses 4 11 Wavelength Settings for SpectraMax Instruments 00 eee eee 4 12 Wavelength Settings for VersaMax Instruments 00 cess ee eens 4 12 Wavelength Settings for Emax Vmax UVmax and ThermoMax Instruments 4 12 SoftMax Pro User s Manual xiii xiv Figure 4 10 Figure 4 11 Figure 4 12 Figure 4 13 Figure 4 14 Figure 4 15 Figure 4 16 Figure 4 17 Figure 4 18 Figure 4 19 Figure 4 20 Figure 4 21 Figure 4 22 Figure 4 23 Figure 4 24 Figure 4 25 Figure 4 26 Figure 4 27 Figure 4 28 Figure 4 29 Figure 4 30 Figure 4 31 Figure 4 32 Figure 4 33 Figure 4 34 Figure 4 35 Figure 4 36 Figure 4 37 Figure 4 38 Figure 4 39 Figure 4 40 Figure 4 41 Figure 4 42 Figure 5 1 Figure 5 2 Figure 5 3 Figure 5 4 Figure 5 5 Figure 5 6 Figure 5 7 Figure 5 8 Figure 5 9 Figure 5 10 Figure 5 11 Figure 5 12 Figure 5 13 Figure 5 14 Figure 5 1
569. sing the baud rate pop up menu in the Preferences The default baud rate shown on the popup menu indicates the current setting for the computer s serial port To reestablish a connection the user must set the serial baud rate of the computer to match the baud rate of FlexStation either 9600 or 57600 other baud rates are included but should not be used 11 11 Chapter 11 Troubleshooting 1 3 11 12 1 2 3 4 5 6 7 port of the computer The A B switch of the switch box is used to physically switch the connection between the instrument and the printer Because of the limitations of the single serial port Macintosh the Autoprint feature of Soft Max Pro cannot be used unless you use the ethernet port of the computer for printing To print from SoftMax Pro for Macintosh using the single serial port Macin tosh connect to an AppleTalk compatible printer such as the Hewlett Pack ard DeskWriter C or 550C using an AppleTalk cable connection and the AppleTalk printer driver Serial printers such as the Apple StyleWriter which use an Apple 8 pin DIN to 8 pin DIN peripheral cable and a serial printer driver cannot be used with SoftMax Pro for Macintosh on a Macintosh with a single serial port Configuration for Reading The computer SoftMax Pro for Macintosh and switch box must be config ured as follows in order to establish communication with the MDC Micro plate System instrument AppleTalk Inactive in the Chooser dialog s
570. sing the Connect Points option the order in which points are connected is sample name dependent 8 63 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments k Axes Options aH Double clicking either graph axis clicking the X axis or Y axis button in the Graph section toolbar or selecting either X axis or Y axis from the Graph menu opens a dialog box concerning the configuration of that axis Figure 8 52 shows the dialog box for the X axis X Axis Settings amp Linear Q Log Figure 8 52 X Axis Dialog Box The default settings are shown in the Figure above You can change these as desired for either or both axes Axis Settings Enabling the Auto range option causes the graph to be drawn such that all data points are shown by automatically adjusting the minimum and maxi mum range settings to encompass the data You can disable the Auto range function by clicking the checkbox or by changing either or both values for the Min and or Max settings Formulas in SoftMax Pro 8 64 You can enter custom calculations in several places within SoftMax Pro to provide the information you desire e In Plate sections you can specify formulas to be used for wavelength reduc tion and Kinetic Spectrum reduction e n the Group table sections you can view and edit the formulas associated with the various columns or create new columns containing new formulas The data shown in columns
571. sing the De lete key on the Macintosh or Backspace key on the PC Select All 88A CTRL A The Select All command can be used in Notes Plate Cuvette and Group sections when a section is active itis not available in Graph sections In a Notes section if no summaries are present it will select all text If summaries are present at the top or bottom of the Notes section this command will select all text but not the summaries which must be se lected individually If the text is broken up by summaries this com mand will select the largest block of text that it can bounded by summaries In a Plate section this command will select all wells if one or more wells in the grid array has already been selected If no wells have been selected this command will be dimmed In a CuvetteSet section this command will select all cuvettes if one or more cuvettes has already been selected If no cuvettes have been se lected this command will be dimmed In a Group section this command will select all columns in the group table but will not include any summaries at the bottom of the group table summaries must be selected individually Delete Section Selection 3 CTRL Deletes the currently active sec tion s or experiment s A warning dialog box will appear asking if you really want to delete the section s or experiment s SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Duplicate Section Experiment
572. sodb d Re pP Re E pu e USO bar piis 7 18 Masking Wells or Cuvettes quesevmns sande ere err ape se 7 22 Pata Reduce BOLYou cio dra nt RS doe VR osea ERE RR 7 23 Plite SO CUNO DL oed dein Send od opis othe gu due heap aus Sha 7 23 C UVECE DECUION ooo a eA OS RM Re UI Me pd eria dd 7 23 EG POM MR ODE 7 27 eiae sina duro od ane nuts o UE ed atnsug pou ubi utu eter 7 28 Spectrum SpectraMax Instruments Only sss 7 36 Data Mode Transmittance Absorbance 7 37 Custom Reduction Formulas 0 0 0 esses 7 38 B calculation DODS 23 cose do ps DPI 7 40 Displaying Data in Group Graph and Notes Sections 7 41 GIOUDIDECLIOHS Des S eque GA ah Ghai Aang Hohe Sana Scene ae 7 41 NOTES DeECBOTISsceossced pee Dawe deo uet eis oe ase 7 47 Graphsechoni uo passi oat d eres Une pide Et 7 47 Formulas in SoftMax PrO 233 4 ARA aces Kraus eoa 7 67 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments t 7 2 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Introduction SoftMax Pro User s Manual Initial display and reduction of the data received from the MDC Microplate System instrument is performed in the Plate section or CuvetteSet section in which the data has been collected Display and reduction parameters can be set up either prior to or after collecting data The display and reduction parameters may be modified at any time Dat
573. specified in Page Setup when a new column is added the columns will disappear off the right side of the window but are still there To bring these columns back into view resize visible columns smaller SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual New columns can contain references to other columns both in the current section or in a different one within the same experiment For example if you wanted to subtract the mean values in one group from those in another you could create a new column in either group to do this If the two groups were named Group 1 and Group 2 for example and both groups contained a column entitled Mean you could create a column within Group 1 that would subtract the mean values in Group 2 from those in Group 1 The column formula needed to do this would be Mean MeanQGroupZ Numerous possibilities exist for creating and editing column formulas to dis play the data you desire For a complete discussion of formulas and opera tors see the accompanying Formula Reference Guide whose To delete a column highlight it and press the Delete key or choose Cut from the Edit menu 8 43 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 8 44 Adding or Editing a Summary in a Group Section To add a summary at the bottom of a Group section click the Cre
574. ss word The administrator has a high degree of responsibility This person will man age the audit trail in case it is necessary to trace issues The administrator manages all SoftMax Pro user permissions including the permission granted to certain individuals to sign E statements which allow for electronic signing of documents to approve verify or authorize them All E signatures made in SoftMax Pro are legally binding provided the customer has the correct operat ing procedures in place Your lab group must understand which permissions will be granted to which SoftMax Pro users These permissions are set in the Enterprise Admin istrator software The permissions that are set allow levels of functionality in SoftMax Pro The administrator performs these tasks e Installs the Enterprise Administrator software e Assumes the role of administrator within the software and create a data base e Adds individual SoftMax Pro users to the database and grant them specific permissions e Installs SoftMax Pro on individual users PCs and link the database file path location to their SoftMax Pro program e Sets the Autosave file path in SoftMax Pro so that data files are saved auto matically in a pre determined location Neg In Windows version 4 3 of SoftMax Pro Enterprise Edition the soft ware preferences are saved in the user s folder of Windows under SoftMax Pro User s Manual Chapter 6 SoftMax Pro Enterprise Edition and Enterprise Ad
575. ssary of Terms Absorbance A The amount of light absorbed by a solution To measure absorbance accu rately it is necessary to eliminate light scatter In the absence of turbidity absorbance optical density reflection A log Io 1 Ip incident light I transmitted light In this manual we use the terms absorbance and optical density interchangeably Active The window or icon that you are currently using or that is currently selected ANSI Character Set The American National Standards Institute 8 bit character set It contains 256 characters Area Under Curve Reduction formula for a Kinetic or Spectrum scan that determines the area under the Kinetic or Spectrum plot or under a plot in a Graph section First the area under the curve is divided into a series of trapezoids then the area defined by each trapezoid is computed and all areas are summed ASCII Character Set The American Standard Code for Information Interchange 7 bit character set It consists of the first 128 0 127 characters of the ANSI character set and most other 8 bit character sets The ASCII character set is the most universal character coding set Author Name The Author Name is entered from the password dialog box at the time when file shielding is turned on The Author Name is different from the User Name in that it may be the name of the author of a protocol file that is later used to read data Appendix B Glossary of Terms AutoC
576. ssay Plate Type Wells to Read AutoRead SoftMax Pro User s Manual One Excitation 485 Emission 538 Auto Cutoff 6 readings Auto PMT 96 Well Standard Read entire plate Off 10 5 Chapter 10 Tutorials L 3 Define the Protocol The following steps will guide you through the process of creating a protocol file for a quantitative assay Reading an actual plate will not be required you will use a simulator to read a plate and generate data Step 1 Start SoftMax Pro Create a New File Double click the SoftMax Pro icon to start the program If SoftMax Pro is already running choose New from the File menu You should see a new file labeled Untitled The figure below shows an example of both the absor bance bottom left and fluorescence upper right Default Protocol file 10 6 SoftMax Pro User s Manual Chapter 10 Tutorials inl NT cs Introduction ia NE Notes Z4 f ia Fluorescence Tutorial 1 Default Protocol Bi Default Protocovrutoriat 1 If you are new to SOFTrax PRO follow this tutorial Tutorial 1 as outlined in Chapter 10 of the SOFTmax PRO User s Manual This protocol contains this Introduction and one Experiment section You can also read Chapter 5 Reading Microplates Using Fluorescence Instruments and Chapter 8 Displaying and Analyzing Data Using Fluorescence Instruments and refer to the Formula Reference Guide to utilize the full potential of SOFTmax PRO Experimen
577. ssible to use a partial rack of tips and to choose which tips are available in the Pipette Tips Layout setting do so with caution ensuring that the lay out described in the software matches the actual tips inserted in the tip drawer Compound amp Tip Columns The choices in this window depend upon the number of transfers chosen in the Compound Transfer tab When one or more transfers are enabled these settings allow you to choose the tips and compounds that will be used for transfers 5 46 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Compound amp Tip Columns SoftMax Pro User s Manual 1 z E 4 Figure 5 48 Compound amp Tip Columns Settings Box SoftMax Pro automatically enters information in the Compound and Tip Col umns tab using a best fit algorithm that uses the number of transfers the Wells to Read section and the Pipette Tips Layout selected area The algo rithm assumes fluid will be aspirated from the compound plate starting with the first available column indicated in the Pipette Tip Layout That fluid will then be dispensed to the first available column indicated in the Wells to Read selection The algorithm assumes e All columns in the Wells to Read selection should receive fluid e The fluids are dispensed from left to right e The fluid transfer targets are cumulative from transfer to transfer that is the second transfer s targets start with the next
578. st If Autoprint is enabled make sure the printer is connected and that it is turned on prior to starting a reading Ifyou are using a Macintosh you can check the Chooser for the currently selected connected printer EH If you are using Windows you can select a printer by choosing Print Setup t from the File menu In the Print Setup dialog box choose the default printer or select from any other printers have been configured for use with Windows See your Windows documentation for further information SoftMax Pro User s Manual 9 11 Chapter 9 File Management and Printing 1 B The Printed Report You can create customized reports by including or excluding experiments and individual sections changing the order in which sections will print and add ing text describing each experiment in Notes sections You can also create summaries consisting of reduction formulas and associated text in Notes and Group sections The size and content of Group sections can also be changed Including or Excluding Sections When sections are created they default to being included in the printed report You can exclude sections from printing by clicking the print setting button on the tool bar of that section it is a toggle so it will change from enabled 8 to disabled z or vice versa Changing the print setting option for the experiment will change the state of all sections within that experiment Changing the print setting option f
579. store box to return the window to its previous size Keyboard users can use the Restore command on the Con trol menu Rotated Display A display option for 384 well plates that shows the plate rotated 90 degrees Scroll To move up down right or left through text or graphics in order to see parts of the file or list that cannot fit on the screen A scroll bar appears at the bot tom and or right edge of a window whose contents are not entirely visible Each scroll bar contains a scroll box and two scroll arrows A scroll box is a small box that shows the position of information currently in the window or list box relative to the contents of the entire window A scroll arrow is an arrow on either end of a scroll bar that you can use to scroll through the con tents of the window or list box Semi Log Curve Fit The semi log function fits the best straight line to a set of data for log X plot ted against Y The resulting curve displayed will be a straight line with the X axis drawn in logarithmic scale Serial Port PC Windows ai A connection on a computer usually COMI where you plug in the cable for a serial device such as a plate reader a printer or a modem Serial asynchro nous and RS232 interfaces are all the same type by which the computer sends single bits of information to the connected device one after the other Win dows supports COMI through COM10 MDC Microplate System instruments require COMI or COM2 Series
580. strument faster than in real time Note that these same values are contained in a file called Endtut pda in the Tutorial folder within the SoftMax Pro folder on your hard drive If you wish you can open and use the data in that file instead from this point for ward The plate section of either file should look similar to the figure below 10 22 SoftMax Pro User s Manual Chapter 10 Tutorials v C3 puer ssa i 7 El Plate 1 B Atomix Off Calibrate On Plate Last Read 12 11 PM 2 400 Pre Read Used Mo Data I nnm c o m zr Wavelength Combination Lr Data Mode Absorbance Figure 10 23 Plate Section with Simulated Data Absorbance instrument example The display for each well that is defined by a group boundary shows a raw value The reduction formula is shown at the bottom of the display Wave length Combination Lm1 A Click the Display button in the Plate section tool bar The Display dialog box will open ws Display E Raw TTE xj Raw OD E Absorbance gt Example a aa ed EN Eawr Reduced Raw RFU 0 330 j Example Peer rere errr reer rere err rrr TI LA ARRRRRRRRRRRRRRRRRRERRRRSRRRRERERRRRRSERRRRARRR Show with reduced number assunussunussuussRESSSENRERENRSHENRSSEESSEER RIEN UN nm With reduced number Raw Data in 1000 s Fluorescence Cancel Figure 10 24 Display Dialog Box The current display selection is shown as a dep
581. subsequent rows or plates starting with the top left point and proceeding with horizontal priority SoftMax Pro User s Manual Appendix A SoftMax Pro Version Specifications Apple OS 9 Navigation Services The methods for finding opening and saving files have changed with OS 9 from Apple Macintosh Navigation Services have been updated in SoftMax Pro to incorporate these changes Compatibility with earlier operating sys tems has been maintained Interprocess Messaging A command for exporting data has been added to Interprocess Messaging The ExportAs command saves files to the Autosave directory or to subdirec tories of the Autosave directory The txt extension is automatically appended to these files Also the GetTiming command for determining the length of time needed to read a plate has been updated to provide more accu rate information Instrument Settings Changes The ability to choose between wavelength and column priority reads has been added to Endpoint and Spectrum modes in the SpectraMax Plus and Spectra Max Plus 9 The ability to use Speed Read for Endpoint reads has been added to the SpectraMax Plus SpectraMax Plus SpectraMax 190 Spectra Max 340PC and VersaMax Password Protection Password protection has been updated to use a new encryption scheme Compatibility with older files has been maintained Minimum Kinetic Interval Minimum Kinetic interval calculations have been updated to inclu
582. symptotes If a sufficient number of points do not define the inflection point and the asymptotes you will receive a no fit error message or an inappro priate fit For best results make sure that the 4 parameter fit is appropriate for your assay and that the entire range of the assay is represented in your stan dards If your standards do not clearly define both a high and low asymptote the spline fit may be more appropriate for your assay Judging a Good Curve Fit SoftMax Pro User s Manual The correlation coefficient is also known as the goodness of fit value because it shows how good a fit exists between the computer curve and the standard data points It measures the degree to which the points fall on the computer curve and may give an indication as to what residuals are to be expected The correlation coefficient is popularly known as R and is between 1 and 1 inclu sive When the data points lie on a perfectly straight line with negative slope then R 1 If the correlation coefficient is not suitably close to 1 or 1 a per fect fit you can simply apply one of the other types of curves without having to reread the plate For nonlinear curve fits SoftMax Pro performs transformations that cause the curve to become more linear The correlation coefficients are derived from 8 61 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments k that transformed data Because of this you
583. t Control Items in this menu allow you to set up and activate MDC Microplate System instrument functions Assays This menu allows you to choose the folder containing the protocols you have saved and shows those protocols below the divider making it easy to open one or more of them A second level of folders can be stored within the Assays folder and will appear on the Assays menu allowing you to sepa rate protocols further as desired The last menu item farthest right on the menu bar changes to reflect the sec tion type that is currently active If a Plate section is active for example the last menu item will be Plate An active Graph section causes this menu to show Graph If an experiment is selected this menu will not be shown Menus function differently depending upon whether you are using Windows or the Macintosh and can function differently depending on the OS in use On the Macintosh clicking and holding the mouse button down while the pointer is on any of the main menu items causes a drop down list to appear To choose a command from a list drag the mouse until the choice is high lighted and then release the mouse button Within Windows and with some versions of the Macintosh OS clicking on the main menu item causes the drop down menu to remain allowing you to click once on any menu item to choose it Additionally a number of buttons are available in different sections of the SoftMax Pro experiment and section tool
584. t 1 Quantitatrve Endpoint assay with Standards and Unknowns with and without dilution factor The unknowns are Absorbance interpolated from a standard curve Tutorial 1 Default Protocol If You Are Familiar with SOFTmax PRO Customize This Default Protocol 1 Delete any sections you do not want through the Edit menu 2 Make any changes you wish to the Instrument Settings by pressing Setup it is assumed you have an instrument connected 3 Click the Template button and assign any default or new groups as necessary you can also select replicates 7 an Introduction Sst NE Motes El Eg 4 Select the desired Reduction and Display Raw vs Reduced parameters 5 Save this default protocol with the name Default Protocol in the sare folder as the SOFTrmax PRO application Default Protocol Tutorial 1 you will be asked if you wish to replace the existing default protocol choose Yes This modified protocol will now launch each time you open SOFTmax PRO A copy of the original default protocol can be found in the If you are new to SOFTrmax PRO follow this tutorial Tutorial 1 as outlined y Tutorial folder if needed User s Manuel This protocol contains thos Introduction and one Experiment sc You can also read Chapter 4 Reading IvDeroplates Using Absorbance Instr 7 CH Experiment inj and Analyzing Data Using Absorbance Instruments and refer to the Formula Vv Plates GH Setup E Template Reduction E
585. t Kinetic Spectrum or Well Scan and read type fluorescence luminescence or time resolved fluorescence The term cut off refers to filters used to condition the light entering or exiting the mono chromators in order to reduce stray light and minimize background interference stimer of wavin E weis l Excitation Emission M Auto Cuttoff unt 9 Jer so ma eos T sso rer Figure 5 11 Cutoff Settings Window The choices you see depend upon the setting for the read mode No emission cutoff filter is used when luminescence is selected With other read types choices are to enable or disable Auto Cutoff with specific filter settings available if it is disabled manual cutoff selection When Auto Cutoff is enabled 5 15 Chapter 5 Reading Using Fluorescence or Luminescence Instruments the instrument sets the cutoffs based upon the wavelength s chosen for reading when Spectrum is selected as the read mode however a manual setting for the emission monochromator is required with the default being no cutoff filter Determining a manual setting for a cutoff filter is done based upon the known value of the Stokes shift the difference between the wavelengths of the excitation and emission maxima If the Stokes shift is small it may be advis able to choose an excitation wavelength that is as far as possible from the emission maximum while still being capable of exciting the fluorophore Doing this causes les
586. t Selected Figure 9 14 Export Dialog Box Windows and Macintosh Exported tab delimited ASCII data files have a minimum of five lines and in most cases will be considerably longer An example of an exported tab delim ited file containing Endpoint data collected from a SpectraMax 250 follows The reading was done at a single wavelength displayed as raw data in a microplate format with labels 0 16487 0 20138 0 24596 0 30042 0 36693 0 44817 0 54739 0 18221 0 22255 0 27183 0 33201 0 40552 0 4953 0 60496 0 20138 0 24596 0 30042 0 36693 0 44817 0 54739 0 66859 0 22255 0 27183 0 33201 0 40552 0 4953 0 60496 0 73891 0 24596 0 30042 0 36693 0 44817 0 54739 0 66859 0 81662 0 27183 0 33201 0 40552 0 4953 0 60496 0 73891 0 9025 0 30042 0 36693 0 44817 0 54739 0 66859 0 81662 0 99742 10 1 405 1 12 96 0 33201 0 36693 0 40552 0 44817 0 4953 0 54739 0 60496 0 66859 0 73891 0 81662 0 9025 0 99742 1 10232 1 21825 SoftMax Pro User s Manual Chapter 9 File Management and Printing SoftMax Pro User s Manual An example of an exported file containing Group table data is shown below BHIBL CKS 1 Group Unknowns Sample Wells Une n1 n Una B1 B Und C1 C2 Unda D1 D Und E1 E Und F1 F2 Und G1 G2 Un H1 H End 1 2 d 1 Sample Values 8 188 6 111 0 122 0 135 0 149 8 165 0 182 4 261 0 223 8 246 0 272 0 300 0 332 Aso 8 46
587. t are available if less than a full rack is used whereas the choices in the menu beneath Fill represent the actual tip numbers that are listed as being avail able in the Pipette Tips Layout tab Similarly to assign a compound highlight one or more compound cells and select one of the items from the Compound pop up menu The wells shown in this tab setting convey graphically the volumes of liquid in the assay plate for each pipetting event Using the color associated with each event the compound settings will display the dispensed compound vol ume as a percentage of the total assay well volume If you entered an initial volume on the Compound Transfer tab setting that will be shown as a gray fill As the liquids are cumulative the first event s volume is shown above the initial liquid volume if any the second event s volume will be shown above the first and the third event s volume above the second To deselect a tip or compound assignment first select the appropriate cell s and then press Backspace on the PC or Delete on the Macintosh To change an assignment select the well s and choose new values or type a value on the keyboard Note that if you have multiple wells selected and you type a value that value will be shown in the first selected well and subsequent wells will increment to the next higher value Again this does NOT take into account the actual pipette tips shown to be available in the Pipette Tips Layout sele
588. t allow you to modify the way in which SoftMax Pro performs recalculation Suspend Recalculation and Recalculate Now 3 CTRL Choosing Suspend Recalculation disables the automatic recalculation feature of the program and places a checkmark to the left of the command in the menu to indicate that it has been selected While this function is enabled no recalculation will occur regardless of what you may change add or delete from the experiment This is especially useful when creating or changing col umn formulas within Group sections for example as it can speed up the pro cess greatly When recalculation has been suspended you may wish to see the results of the changes you have made but may still not wish to enable continuous recal culation Choosing Recalculate Now 3 CTRL from the Edit menu will cause SoftMax Pro to examine all elements of the experiment and perform complete recalculation of the data Choosing this command does not enable continuous recalculation but simply causes the program to perform recalcula tion once 7 40 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments To return to continuous recalculation choose Suspend Recalculation again The checkmark next to the command will disappear indicating that the function is no longer in use NOINA Recalculate Now 3 CTRL can be used to see intermediate results during long Kinetic readings At any time
589. t settings are for an Endpoint reading or if no well in the Plate display has been selected Button Equivalent in the Plate section tool bar Mask Allows you to hide certain wells so that they will not be taken into account when performing analyses This command is dimmed if no SoftMax Pro User s Manual 3 33 Chapter 3 SoftMax Pro Overview 3 34 data has been acquired or if no wells in the plate have been selected Button Equivalent in the Plate section tool bar Copy Template While a Plate section is open and active choosing this com mand allows you to copy the template from this Plate section to the Clipboard allowing you to paste it into a different Plate section in the same or in a different experiment or into a Plate section in another SoftMax Pro file Paste Template If you have copied a template from a Plate section you can then create or activate open another Plate section and choose Paste Template In this way you can use the same template for more than one Plate section or you can modify the pasted template as desired If you are pasting a template into a new experiment and groups from the source template do not exist in the destination experiment pasting the template will create them Existing groups of the same name will not be changed by the pasting process For example if the template be ing copied contains the groups Standards and Unknown and the group Standards already exist
590. tMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Point to Point A linear equation is fit to each pair of data points Fit parameters are not given The point to point curve fit is a linear fit composed of pieces that assume a linear relationship between each pair of data points The line seg ment defined by each pair is used to interpolate data between those points Since there are multiple line segments fit parameters are not shown on the graph 2 Graphis Fit Point Ta point Graphz3 B Concentration Floti Groupzz Concentration vs Values Figure 8 46 Graph with Point to Point Fit Cubic Spline This curve fit generates a fit to a cubic equation between each pair of data points The general form of a cubic equation is y A Bx Cx Dx The equations are computed with the requirement that the first and second derivatives of the equations are continuous throughout the range of the data As multiple curve fits are performed with this routine fit parameters are not shown with the graph v Z Groen Fi eec T Graphz4 B concentration Plot 1 Groupe Concentration ve Mean alue Figure 8 47 Graph with Cubic Spline Fit SoftMax Pro User s Manual 8 59 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 8 60 Exponential The exponential function used to generate this curve
591. ta Figure 10 56 The Plate Section Showing Group Boundaries 10 50 SoftMax Pro User s Manual 3 Chapter 10 Tutorials Step 4 Set the Reduction Parameters Click the Reduction button in the Plate section tool bar to open the Reduc tion dialog box The default reduction setting for a single measurement wave length at the top of the dialog box Lm1 is acceptable Beneath this is a choice for Kinetic Reduction Here the default choice should be changed Click in the drop down menu and choose Max Min as shown in the illus tration below miReduction E X e Wavelength Combinatiorr UE lal col clei cael Sea dea dure d ahead cae yl ye yd wil wi ly yl oll yl d lol loc fc lc bool oe le gel ee veel lad lg aed wad aed shod ase sated cated ase uod yo ye lyre oytenih E w VIMax units per sec Time to Max Onset Time Absolute Time at Minimum Tic HESSE irme 2 Zero Baseline Points 7 Time at 1 2 Maximum i C Baseline Multiplier Peak Max Min Slope Area under Curve Mean Custom Figure 10 57 Reduction Dialog Box The other values in this dialog box should be set as follows see Figure 10 58 e Limits Min RFU 0 Max RFU 50000 Lag Time 0 End Time 200 e Display Options Zero Baseline 3 Pts e Smoothing Moving Average 1 point no smoothing SoftMax Pro User s Manual 10 51 Chapter 10 Tutorials 3 m Reduction x DEN Wavelength Combinatior ee te ae
592. ta0l s iw Top EJ Sample Descriptor E OBottom eman vts pa Rx 5 O Left Starting value Replicates 3j Figure 4 28 Series Dialog Box 3 Specify the name of the first sample in the series in the box or accept the default provided by SoftMax Pro Subsequent replicates use this name as a base and either increment the number within the name or append a number to it In the example above the sample name provided by Soft Max Pro describes the first sample 01 in a group named Standard here shortened to Sta 4 Theinformation shown in the box labeled Sample Descriptor is derived from the information entered in the Group Settings dialog box If the in formation for sample descriptor or units is not correct you may change it here If you did not already do so specify the starting value for the first sample in the series Values for subsequent samples are calculated from this starting value 5 Choose the operator for the series or from the Step by drop down list Enter the starting value for the series in the Starting value box The increment is entered in the box to the right of the Step by box and below the Starting value box Figure 4 28 shows a starting value of 5 with the operator being division and the increment being 2 Thus the series will start with a value of 5 and divide each subsequent sample by 2 to produce the following series values 5 25 125 etc 6 You
593. tart wavelength Spectrum start wavelength otherwise blank field End wavelength Spectrum end wavelength otherwise blank field LM Wavelength step Spectrum wavelength step otherwise blank field of wavelengths of wavelengths in Endpoint Kinetic Well Scan FLEX reads for Spec trum blank field Read wavelengths For absorbance luminescence wavelengths read separated by a space for Spectrum blank field for fluorescence emission wavelengths for Ex Em sweep blank field La First column read First column read 1 to max always 1 for cuvettes 18 Number of columns of columns Last column read 1 to max Last column read 1 to max always 12forcuvettes 12 for cuvettes a oo wells or Number of wells in a plate PLE ALIE ae E ce MENU 96 384 etc or number of cuvettes read cuvettes for a CuvetteSet After Field 19 the format splits depending on the read type Fields 20 28 are not present for absorbance or transmittance but are included for fluorescence and or luminescence 20 Excitation wave Excitation wavelengths separated by spaces lengths For luminescence or sweeps this is a blank field the emSweep excita tion wavelength is in field 16 m Reads per well Number of times a wellis read fora single reading 26 PMT setting PMT setting Automatic Automatic High Medium or Low Medium or Low z Start integration Time to start integration for time resolved fluorescence Anoma
594. te reduction parameters and display parameters Prepare the microplate s to be read and place them in the instrument If using a FlexStation also prepare any compounds to be used and load the pipette tips Initiate the reading Save the data file Modify the template reduction parameters or display parameters as you wish or if you didn t set them up in Step 4 do so now Save the data file Analyze the data using the Group section tables and Graph sections Save the data file when you are finished This chapter discusses how to define instrument settings create a template and initiate a reading using fluorescence or luminescence instruments SoftMax Pro User s Manual 5 3 Chapter 5 Reading Using Fluorescence or Luminescence Instruments Instrument Settings NOJA Only the settings that pertain to the instrument that is connected to the computer or the one chosen in the Preferences will be shown in the Instrument Settings dialog box You must specify the instrument settings prior to actually reading a micro plate These settings include e The read mode Endpoint Kinetic Spectrum Well Scan or FLEX mode e Wavelength s used with Endpoint Kinetic and Well Scan modes or the wavelength range for Spectrum mode e Timing for Kinetic and FLEX mode runs e Cutoff filters e PMT sensitivity and number of readings e Read type fluorescence luminescence or time resolved fluorescence for most instruments fluo
595. te Database Dialog DOXi ta es Pad eio egre mi ed Aq EARE AREA a dos 6 8 Database Save As Dialog Box 6 cee e 6 9 Database Creation Messages cu2x 03 26e ribs vsus es dares tendu a d Pru eed neaei 6 9 Administrator Login Dialog Box 1 6 ee cee ee 6 9 Administrator s Dialog Box Users Tab shown 00 ee ee ee eens 6 10 epp 4r e eee pees 6 11 REPO LACS P E AE AA En reee 6 12 Eleac An PENE EE EEEE 6 13 User Information Page in Add New User Wizard 0 00006 6 14 User Privileges Page in Add New User Wizard lsleeeel eese 6 15 User Privileges Dialog DOXLe ve tesque quami ed omded ries 94 dudit idi de ads 6 16 Modity User Dialog DOX s aux eux dan isa etie tore Rr RERO E PLE SUEDE attri 6 18 Remove User Warning Message a ideae ich e OPAC SCPARUR A qn dc vedo goa ses 6 18 Add Software Product Dialog Box 0 cee cee een 6 19 Delete 5otbware Dialog BOX o4 tpud S 43m Siu rea e desde dard d Rcs 6 20 SoftMax Pro User s Manual XU xvui Figure 6 16 Figure 6 17 Figure 6 18 Figure 6 19 Figure 6 20 Figure 6 21 Figure 6 22 Figure 6 23 Figure 6 24 Figure 6 25 Figure 6 26 Figure 6 27 Figure 6 28 Figure 6 29 Figure 6 30 Figure 6 31 Figure 7 1 Figure 7 2 Figure 7 3 Figure 7 4 Figure 7 5 Figure 7 6 Figure 7 7 Figure 7 8 Figure 7 9 Figure 7 10 Figure 7 11 Figure 7 12 Figure 7 13 Figure 7 14 Figure 7 15 Figure 7 16 Fig
596. ter dividing line The files that appear here are protocol files that reside within the folder determined using the Set Folder com mand You can quickly open and use one of these protocol files by se lecting it from the Assays menu SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Plate Menu This menu item changes to show CuvetteSet Plate Notes Graph or Group depending on the type of section that is currently active If no section is active Template this menu item does not appear Reduction Display Template Opens the Template Editor Graph The Template Editor allows you to describe the samples that are in the Mask wells thus providing the link between the raw data and the analysis groups You can create new groups in the template or assign wells to already established groups Copy Template laste lrempiate If your experiment contains multiple Plate sections the template for each Plate section must be defined using the Template Editor in that section You can create a new template or modify an existing one using section Name the Template Editor SENI QW Each Plate section requires that a template be created in order to identify groups within that plate However two or more Plate sec tions may use the same template or the same groups may be used in different templates Expart Template Import Template After groups are defined in the template and you return to
597. ter the start of data collection Thus the linear curve fit is not good and the slope of the line does not reflect the maximal rate In the figure on the bottom right a lag time of 80 seconds has been set and the linear curve fit for both wells is good allowing the slope of the lines to reflect the maximal rate in each well Alternatively you could leave the Lag Time and decrease the number of Vmax Points used to determine the slope of the line End Time This setting specifies the time at which you wish to stop showing data in the display Any values occurring after this limit will not be reported in the dis play The default setting is the total assay time The value for end time may not be greater than the total assay time 8 23 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Absolute Values This setting causes the plots shown in the data display to be drawn at their absolute RFU RLU as opposed to the default with the box unchecked which offsets the first point to the 0 0 coordinate Enabling Absolute Values may cause some data points that do not fall within the default limits for the plots to disappear see Figure 8 16 mi Reduction E x Wavelength Combinatiorr i um vw N f Kinetic Reduction Eet0000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
598. termined through RegisterWindowMessage This ID ie later used to discriminate between WM_COPYDATA messages received to understand which WM_COPYDATA coming back hae the instrument status unsigned int gExternalKeplyMsglD Entry point for typical MFC windows program int APIENTRY WinMain HINSTANCE hlnetance HINSTANCE hFrevlnetance 12 15 Chapter 12 SoftMax Pro Remote Command Language LPSTR IpCmaLine int nCmdShow Ferform this initialization early in the program gExternalReplyMsg D RegisterWindowMessage SOFTMaxProReplyMsg Callback for processing windows messages received LRESULT CALLBACK WndFroc HWND hWnd UINT message WFARAM wFaram LPARAM IFaram t begin processing incoming window messages switch message af case WM COFYDATA Windows WM COFYDATA message received Check if this message contains status message expected COFYDAIASIRUCT ca cd COPYDATASTRUCT IFaram if cd gt dwData gExternalReplyMsglD length of status message string plus 1 unsigned int len cd cbData instrument status message returned char str char cd lpData Process return data ae you wish 12 16 SoftMax Pro User s Manual Chapter 12 SoftMax Pro Remote Command Language case WM_COMMAND Windows command to process the application menu selection wmld LOWORD wFaram wmEvent HIWORD wFaram Farse the menu selections switch wmld
599. ters the wavelengths for these filters are entered differently using the instru ment front panel For additional information regarding the installation of optional filters in the SpectraMax 250 refer to the instrument man ual Autosave Enabling the Autosave feature in the Preferences dialog box will save a data file automatically each time a plate is read ff Autosave WU E E cece ete m u fs Append Date File Prefix kd Append Time i SOFImax PRO File Text File LuuERERRRRRERERERERRERERRERRRRERRERERERERERRRRRRERRERRRERERRERRRRRERERERERRERRRERRRRRRRERERERERERRERRRRRERERRRRRRERRERRRERRERERERERRHRRRRERREERRRRRERERRERERRERERERERERRERRRRRRRR Figure 2 13 Autosave portion of the Preferences Dialog Box Autosave saves a copy of the file that is currently open If the file that is open is a protocol file note that the file will still be Untitled i e not yet named SoftMax Pro User s Manual 2 19 Chapter 2 Installation even after an autosave has occurred This allows you to autosave to a text file format and then save manually as a SoftMax Pro file You can Autosave to one of two different file formats e SoftMax Pro used to save your data automatically in SoftMax Pro format e Text File used when you want to have the data available for use with a spreadsheet or other program for data analysis external to SoftMax Pro If you autosave as a text file the SoftMax Pro version of the data WILL NOT BE SAVED AUTOMATIC
600. ters 7 and 8 describe how data is displayed during and after a reading and how you can customize the display using absorbance instruments and the fluorescence instruments respectively They also describe data reduc tion and the different means for analyzing the raw data received from the different instruments Chapter 9 File Management and Printing explains how to customize reports and manage the files created by SoftMax Pro Other topics include importing exporting copying pasting duplicating data both within Soft Max Pro and to and from other external programs and details about how to import FLIPR and Analyst System files Chapter 10 Tutorials provides two written tutorials that will help you learn the basics of working with SoftMax Pro if you are a new user Chapter 11 Troubleshooting describes alert messages that may appear while using SoftMax Pro and what to do should they occur Other informa tion in this chapter discusses possible communication problems with the MDC Microplate System instrument and other computer related issues Chapter 12 SoftMax Pro Remote Command Language describes the robotic integration commands that can be used with Windows compatible SoftMax Pro User s Manual Chapter 1 Welcome to SoftMax Pro PCs to control the various functions of the instruments that work with Soft Max Pro e Appendix A SoftMax Pro Version Specifications lists the changes that have been made fo
601. th Dilution Made into a Series I The template is now complete Click the button to accept the entries and close the Template Editor The outlines of the groups you have defined in the Template Editor will now be shown in the Plate section 10 18 SoftMax Pro User s Manual Chapter 10 Tutorials v E3 Patet i5 Plate 1 1 2 3 4 5 B 7 E 9 10 11 12 Lint 405 Automix Off Calibrate On Plate Last Read Wavelength Combination Lm1 Data Made Absorbance Figure 10 17 The Plate Section Showing Group Boundaries Step 4 Set the Reduction Parameters Click the Reduction button in the Plate section tool bar to open the Reduc tion dialog box my Reduction X a Wavelength Combination Absorbance oo Data Mode 1 Use plate blank v Absorbance Use pre read plate Fluorescence amp Tranzmittance j Switch normal and pre read m Reduction GS Wavelength Combination CORR abr x mare tas fetta ite notated teste et ton land ded dar ade a etter PD M Aer ed dates LU ett ed iad asta TET Acer tod T D RU Cancel Figure 10 18 Reduction Dialog Box Single Waveleneth Endpoint The default reduction settings for a single measurement wavelength Lm1 are acceptable and no changes are required For absorbance instruments make sure the radio button next to Absorbance is chosen and that the checkboxes next to next to the options for using a plate blank etc are not ch
602. th their sub sec tions is saved to a separate file Open 3880 CTRL O Opens an existing SoftMax Pro file or an older SoftMax file When you choose Open from the File menu a dialog box appears To open a file from the list of available files double click the filename to be opened or click once on the filename and then click the button Close W CTRL W Closes the front most active window If you have only one file open this file will be closed If you have changed the file in any way since it was opened a dialog box will open asking if you want to save the file before closing This menu item is dimmed if no win dow is open Save 9 CTRL S Saves the active window to a file The active file name is shown at the top of the status window protocol files are always shown as Untitled To save the file in a different folder subdirecto ry or under a different name use Save As If the file has not yet been saved and given a name is Untitled the Save As dialog box will appear and you will be asked to name the file This menu item is dimmed if the file has already been saved and no changes have been made Save As Saves a file with a new file name or into a new folder or subdirec tory See Saving Files Manually on page 9 8 for more information about using the Save As command for saving files Import Export A submenu offers the following choices Import Export Import Criterion
603. the RFU RLU at item X in the list of readings For example if you have a Kinetic run with 20 time points and X is 10 it will report the RFU RLU of the 10th time point Similarly if you have a Spectrum scan with 20 measurements and X is 10 it will report the RFU RLU of the 10th wavelength measured A list of accessors and operators that may be used in custom reduction formu las can be found in the Formula Reference Guide When you finish your entry in the Calculation dialog box and click OK the formula you have entered then appears to the right of the word Custom and becomes the default selection for the Custom option You may subse quently edit this formula or enter a new one by clicking the Formula button again Recalculation Options By default SoftMax Pro performs continuous recalculation of the data when you read plates create or change formulas and so on At certain times it would be useful to disable the recalculation feature so that you can make alterations to the experiment without waiting for individual recalculations to be completed Two commands are available in the Edit menu that allow you to modify the way in which SoftMax Pro performs recalculation Suspend Recalculation and Recalculate Now 3 CTRL SoftMax Pro User s Manual 8 37 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 1 k Choosing Suspend Recalculation disables the automatic recalculation feat
604. the basics of using SoftMax Pro Sample files that demon strate the advanced use capabilities of SoftMax Pro are explained in Chapter 5 of the Formula Reference Guide What s New in SoftMax Pro Version 4 3 Separate Editions and Enterprise Administrator 1 4 Three different editions of SoftMax Pro are now available e Life Science LS Edition for life science use shipped with all bench top microplate readers e Drug Discovery DD Edition for drug discovery data analysis shipped with FlexStation can be used to analyze data files from Analyst systems and FLIPR systems Enterprise Edition EE for customers who require 21 CFR Part 11 compli ance features contains all features from both the Life Science and Drug Dis covery editions as well as new compliance features The Enterprise Edition of SoftMax Pro links with a separate software package Enterprise Adminis trator through a menu choice in SoftMax Pro SoftMax Pro User s Manual Chapter 1 Welcome to SoftMax Pro 21 CFR Part 11 Compliance New File Extensions for Enterprise Edition User Access in Enterprise Edition SoftMax Pro User s Manual e Enterprise Administrator EA software is a separate software module for Windows only that links with the The Enterprise Edition of SoftMax Pro Enterprise Administrator provides the capability to assign multiple users up to 175 and multiple permissions privileges and electronic signature capability e
605. the cus tom groups that have been created by default or by the user The figure below shows two different instances of the appearance of the Group list SoftMax Pro User s Manual 4 23 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments Patients Standards my Unknowns Blank Mew No custom Example of custom groups created groups created by a user Figure 4 19 The Group Drop Down List Depending on the default template used when opening SoftMax Pro initially no groups may appear above the gray line As you create new groups they will be positioned above the line e Blank Creates a plate blank in the Plate section or a Template blank in the CuvetteSet section Note the name blank is reserved for use by the Soft Max Pro program and should not be used when creating custom groups e New Creates a new group The Group Settings dialog box will appear see Figure 4 20 showing default settings This dialog box allows you to define the name for a group of related samples what type of descriptor will be associated with these samples and the initial column format for the Group section Whenever a new group is created whether or not wells are selected in the template a Group section table will also be created To delete a group you must delete the Group section Simply clearing a group from the Template Editor will remove the assignment of wells to that group name but the name will still sho
606. the fit Figure 7 53 below shows a scatter graph with points connected compared to a linear fit NOIA The Connect Points option connects the points in order of appear ance by sample name not the appearance of points from left to right on the graph SoftMax Pro User s Manual 7 65 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 66 Graph Options Button d s f X and Y Axis Buttons concentration y A Bx A Plot Groupz1 concentration vs Mean y alue Figure 7 53 Scatter Graph with Points Connected Linear Fit Applied Other options in this dialog box allow you to plot the data as a bar graph with data shown in clusters or stacked Figure 7 54 shows both of these bar graph options Fit Nort v Graph 1 7 Graphit Graph 2 0 4 5 Sample Number E Plot 2 Group 2 Samples vs Mean value E Plot 1 Group 1 Samples vs Mean alue eee mmm A A A Plot 3 Group 3 Sample vs Mean alue Cluster Bar Graph p EQ A A RN DIL aaae Stack Bar Graph Sample Number BE Plot 1 Group 1 Samples vs Mean value Plot 2 Group 2 Sample vs Mean value A Plot 3 Group 3 Samples vs Mean alue Figure 7 54 Bar Graphs Cluster and Stack NOFA Dar graph options for multiple plot graphs are only available when eac
607. the height of the pipette tip above the bottom of the well of the assay plate It is not necessary to perform trituration during the Tutorial assay however so ensure that neither box for Compound Source or Assay Plate is checked and proceed to the next item Pipette lips Layout SoftMax Pro User s Manual ANISNSEGSR Molecular Devices recommends that you use a full rack of tips each time you perform a transfer of fluid in FLEX mode since this prevents potentially serious errors from occurring when partial tip racks are used For example if you should mistakenly enable a pipet ting function from a tip that is not present or if you enable more or fewer tips than are actually available the instrument can malfunc tion potentially causing serious damage While it is possible to use a partial rack of tips and to choose which tips are available in the Pipette Tips Layout setting do so with caution ensuring that the lay out described in the software matches the actual tips inserted in the tip drawer This setting allows you to set the location of the pipette tips that will be used for the assay For this tutorial a full set of tips will be used the default so no change in settings is required 10 45 Chapter 10 Tutorials M Instrument Settings ol al el ol fe Endpoint Kinetic Spectrum Well Scan Options Before Off Between Off AutoCalibrate Once Assay Plate Type 96 Well Standard clrbtm Wells To Read Re
608. the program default enables sub traction of a background constant default is 0 0 from the absorbance values that are read Often users do not take the time to pre read plates which pro vides the most accurate method for reducing background signal from absor bance readings Subtracting a background offset provides a better result if no pre read blanking is chosen NOJA If you choose to pre read a plate be sure to disable the background constant subtraction both choices are available in the Instrument Set tings dialog box To pre read a plate place water in the wells Wells pre read with water gener ally have less absorbance than do dry wells e g at 426 nm Nunc microplates read with and without water in the wells give absorbance values of approxi mately 0 037 and 0 049 respectively Thus pre reading a dry plate results in pathlength corrected absorbance values that are lower than the cuvette val ues If the pre read step is omitted entirely the pathlength corrected results are overcorrected If the plates are sufficiently transparent at the wavelength s of interest and have low well to well variability the pre read doesn t have to be made on the same plate that will contain samples another clean plate from the same lot will be adequate More conveniently a single plate can be pre read and that pre read data can be stored for subsequent use SoftMax Pro allows you to copy and paste pre read plate data from one plate section
609. the wells on the source plate same group and sample names Any changes made to the template on the destination plate will also be made on the source plate Data from the two Plate sections CuvetteSet sections will be analyzed together Copying and Pasting between Different Experiment Sections To copy a template make the section containing that template active and choose Copy Template from either the Plate or CuvetteSet menu the menu name changes to reflect the type of active section Then activate the destina tion Plate section or CuvetteSet section and choose Paste Template from the Plate or CuvetteSet menu The group names and sample names of the pasted template will be identical to those of the source template but since they are in different experiments the full name will be different for example group experiment 2 instead of group experiment 1 Any changes made to the template of the destination Plate section CuvetteSet section will not affect the source section Data from the two Plate sections CuvetteSet sections will not be analyzed together Copying and Pasting Template Regions Contiguous regions of templates can be copied and pasted to other regions of templates within the same or to a different experiment All template settings are pasted into the new template except the group name the new group is given the name Group X where X is the next greater number than that used already for group names To copy a region of
610. they are grouped single wells are still selectable in either the Template Editor or the data display Samples and groups may exist across multiple Plate sections as well as within a single Plate section Each template is built upon a hierarchy as follows the template is composed of samples and groups A set of one or more replicate wells makes up a sam ple A set of related samples forms a group For example you may have a group named Standards that consists of seven samples named STD01 STD02 STD07 Group names may be up to 32 characters long while sample names may be 300 characters long Samples may be assigned one at a time using the Assign button or several at the same time using the Series button If you create a group in the Template Editor but do not assign any wells to the group a section for that group will still be created automatically when you close the template You can remove the group both its section in the window and its group name in the template by making that Group section active and choosing Delete Groupname where Groupname is the name assigned to that group in the template from the Edit menu Using the Macintosh you can also delete the selected Group by pressing 3 within Windows you can press CTRL Note that deleting the wells assigned to a group from the tem plate does not delete the corresponding Group name from the drop down list The only way to remove a group name from this list in the Templ
611. time at the maximum sig nal within the reduction limits Time at Minimum Reduction for a Kinetic or FLEX run that reports the time at the minimum sig nal within the reduction limits Time Resolved Fluorescence Most fluorescence substances are not suitable for this type of reading How ever the fluorescence emitted by lanthanide dyes is delayed long enough to measure fluorescence after the lamp is turned off Time resolved fluorescence is used to reduce the amount of background noise which interferes with fluo rescence The excitation lamp flashes and after it is off the delayed emission is collected for a set period of time before the lamp flashes again Time to Vmax The elapsed time until the maximum reaction rate of a Kinetic or FLEX read ing is reached reported in seconds Title Bar The horizontal bar at the top of a window that contains the title of the win dow or dialog box On many windows within the Windows environment the title bar also contains the Control menu box and Maximize and Minimize buttons On the Macintosh the title bar also contains the Close box and Zoom box Transmittance T The ratio of transmitted light to the incident light for absorbance readings T 1 19 x 100 Triturate FlexStation only To mix a solution by means of aspirate and dispense cycles Undo Cancels the previous operation if possible The paste function is enabled by choosing Undo from the File menu pressing 3
612. ting a new Graph section the Graph Options dialog box opens after choosing either Or in the Plot X dialog The new Graph section is not created however until you click in the Graph Options dialog box SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Graph Options Name of the plot Fonts options you can change this Beni neinte OP ype onts lt Chart options box MO Scatter sin Sample Text i Itie Buc r1 Cluster bar s Sample Text hil Stack bar Legend Delete plot button dA enses bU Plot symbols New plot button X and Y axis Pew J Baie c labels Edit plot button i p Plot 5 Concentration Standards MeanaluedmeStandard Plot name and symbol a X and Y error bar tes ee description Figure 8 32 Graph Options Dialog Box Creating Multiple Plots SoftMax Pro makes it possible to create multiple plots within a graph Each plot will represent data from a group that has been created in the Template Editor or data from a summary or custom formula You may plot data from any group or from several groups on a graph even if the groups are in sepa rate experiments It is possible for example to plot multiple dilution series of an unknown to graph together a series of patient samples or to plot several controls that have been run over time Following is asummary of the steps required to create multipl
613. tion Window Notes Section Tool Bar Results of Tests Performed Ihis fest was run In Date and time this test was run Body Vertied by Accepiabiltv ciena fortis test Optical Density qreater than 0 003 and less than 0 003 Results of Test 1 Acceptable Summaries Results of Test 2 Acceptable Results of Test 3 Acceptable If the tests repart Out of specification please view the individual plate sections to determine which wells are aut of acceptable range Figure 3 10 Example Notes Section Named Results SoftMax Pro User s Manual 3 13 Chapter 3 SoftMax Pro Overview l F The Notes section is used to record information that pertains to the experi ment You can enter text of any kind and add summaries containing reduction formulas The Notes section is divided into two areas e The tool bar e The body You may create multiple Notes sections within the same experiment The tool bar contains the following items shown with their tool bar or mouse equivalents These menu items and buttons are active only when a Notes sec tion is active Table 3 1 Notes Section Menu Commands and Tool Bar Buttons Menu Command Mouse Equivalent ae h j Notes section icon PPPO e oes secum m double click in a new window ia Introducti Name of Notes section Opens the Section dia oo log box double click New Window in the Opens the section in a Notes menu new window Section Name in the Opens the Section Name Not
614. tion with the exported data SoftMax Pro User s Manual Chapter 9 File Management and Printing meu 3 Click to close the dialog box Nesey The preference settings chosen for Export Format also affect how data is copied pasted and Autosaved to ASCII text 4 Display options chosen for the Plate section or CuvetteSet section Whether raw or reduced data will be exported depends upon the display options If raw data is displayed raw data will be exported if reduced data is displayed this reduced data will be exported Open the Plate section or CuvetteSet section from which you will be exporting data Click the Display button in the tool bar of the Plate section or CuvetteSet section to access the Display dialog box Choose the elements of the display set tings that match the data you would like to export raw or reduced data with or without number and so on When finished selecting display preferences click to close the dialog box gm Display z Raw Reduced Vmax units per sec Lm1 Example Options pone High Display Threshold w Low Show With reduced number Cancel Figure 9 13 Display Dialog Box When exporting data as tab delimited ASCII text all reduced dis plays will be exported as numbers regardless of how the data is shown in SoftMax Pro Choosing Grayscale Ranged or Threshold will export the same information as the reduced number display Choosing with number will also
615. tions Template Blanking occurs when cuvettes are assigned to a Blank group in the Template Editor of the CuvetteSet section The average value of all the cuvettes in a tem plate blank group will be subtracted from individual cuvette readings For Kinetic and Spectrum data the blank value is averaged and subtracted on a point by point basis Template blanks are subtracted after the reference reading if any is subtracted Ose Template blanks are only subtracted from cuvettes within the same CuvetteSet section Group Associated Blanks Plates and Cuvettes Blank wells or cuvettes can be assigned within any group other than the Blank group in the Template Editor The average reduced value of the blank wells or cuvettes within that group will be subtracted from individual reduced values within that group only This blanking function uses the reduced number exam ple Lm1 Lm2 Vmax etc only it does not subtract on a point by point basis When a group blank has been assigned it is automatically subtracted To see the reduced number without the group blank subtracted mask the group blank wells or cuvettes Group blanks apply to all wells or cuvettes in a group and thus may be sub tracted from wells or cuvettes in more than one Plate section or CuvetteSet section Blanks in Combination It is possible to use blanking functions in combination You could perform a pre read plate blanking assign a blank group in the template an
616. tive and choose Copy Template from either the Plate or CuvetteSet menu the menu name changes to reflect the type of active section Then activate the destina tion Plate section or CuvetteSet section and choose Paste Template from the Plate or CuvetteSet menu 4 35 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 4 36 Template Reduction Template Reduction Cuvetteset New Cuvette tek toe Help New Cuvette Display Graph Display Graph kdack Mask Template Reduction Template Reduction Mask Display Graph Mask Display Graph Mask Copy Template Paste Template Copy Template Copy Template Paste Template Export Template Import Template Export Template Import Template Copy Template Paste Template Section Name New Window Section Name Mew Window Export Template Import Template Export Template Import Template Section Name New Window Section Name Sew Window Figure 4 32 Copy Template and Paste Template Commands in the Plate and CuvetteSet Menus The group names and sample names of the pasted template are identical to those of the source template In effect the destination template is considered an extension of the original template and all wells on the destination plate are considered replicates of
617. to another pro vided the instrument settings are identical in the two plate sections If the plates are inherently variable at the wavelength s of interest however it may be necessary to use the same plate for pre reading and reading samples to ensure that adequate well by well correction is achieved If you are using a SpectraMax Plus or Plus and PathCheck is enabled you can choose to Read Cuvette Reference or to use the Water Constant Read Cuvette Reference requires that you place a standard 1 cm cuvette containing water or aqueous buffer in the cuvette port The instrument then takes readings of the cuvette at 900 and 1000 nm and uses those values for pathlength correction Water Constant uses parameters stored in non vola tile instrument memory at the factory based on readings of deionized water The SpectraMax 190 and 340PC also use the water constant when perform ing pathlength correction 4 15 Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments 1 k To determine a number for use as a background constant pre read an entire microplate at the wavelengths of interest Average these numbers and use the average as the background constant PathCheck on SpectraMax Plus Plus 9 Cuvette Reference or Water Constant Which Should I Use The PathCheck measurement is based on the absorbance of water at 1000 and 900 nm If the sample is completely aqueous has no turbidity has a low salt c
618. toff 8 455 9 355 110 255 11 355 12 455 13 755 15 155 16 755 18 455 20 255 22 455 wavelength Combination ILm1 10 255 11 255 12 455 13 755 15 155 16 755 18 455 20 355 22 455 24 855 27 455 12 455 13 755 15 155 16 755 18 455 20 355 22 455 24 855 27 4553 30 255 33 455 15 155 16 755 18 455 20 255 22 455 24 855 27 455 30 255 22 455 26 855 40 755 v Plate E Template E Display em 18 455 20 355 22 455 24 855 27 455 50 255 53 455 36 855 40 755 45 055 49 755 i T OO mo m oO 60 O cx Fluorescence Raw data in 1000 s Plate 1 22 455 24 855 27 455 30 255 33 455 26 855 40 755 45 055 49 755 54 855 60 655 o H 3 955 4 355 4 755 5 255 5 755 6 355 6 955 7 655 8 455 9 355 4 755 5 255 5 755 6 355 6 955 7 655 8 455 9 355 10 255 1 1 355 5 755 6 355 6 955 7 655 8 455 9 355 10 255 1 1 355 1 2 455 13 755 6 955 7 655 8 455 9 355 10 255 11 355 12 455 13 755 15 155 16 755 8 455 9 355 10 255 11 355 12 455 13 75515 155 16 755 18 455 20 355 10 2551 1 355 1 2 455 1 3 755 1 5 155 1 6 755 1 8 455 20 355 22 455 24 855 12 455 1 3 755 15 155 16 755 18 455 20 355 22 455 24 955 27 455 30 255 4 cC OO mo m oO 0 wo F 15 195 16 755 18 45520 355 22 455 24 855 27 455 30 255 32 455 36 855 19 455 20 259 22 455 24 852327 495 30 255 33 453 36 855 40 755 45 095 Vertical Display
619. tor Dilution Factor y D EL Standards Standards Stali 1 ego Concentration j Unknowns Unknowns Dilution Ungi 1 ug ml Dilution Factor 10 Unknowns Unknowns Dilution Unt 1 ug ml Dilution Factor 10 Unknowns Unknowns Dilution UnD1 1 ug ml Dilution Factor 10 Unknowns Unknowns Dilution Undo 1 ug ml Dilution Factor 10 Unknowns Unknowns Dilution Uno 1 ug ml Dilution Factor 10 Unknowns Unknowns Dilution Un09 1 ug ml Dilution Factor 10 Unknowns Unknowns Dilution Und F 1 ug ml Dilution Factor 10 Unknowns Unknowns Dilution Unt 1 ug ral Dilution Factor 10 Unknowns Unknowns Dilution Und 1 ug ml Dilution Factor 10 Blank Empty EL D Standards Standards Stali 1 g ml Concentration Unknowns Unknowns Dilution Uno2 1 ug ral Dilution Facto 2 2222222z Figure 4 36 Column Data in the Exported Template Information Examining the exported information from your individual template files will help you determine the best method for template creation outside of SoftMax Pro in order for the data to be acquired in the correct manner Reading a Microplate or a Cuvette SoftMax Pro User s Manual When you read a microplate using any MDC Microplate System instrument or when you read a cuvette using the SpectraMax Plus or Plus the informa tion sent by the instrument to SoftMax Pro is absorbance optical density data Optical density is defined as log
620. torial is designed to be used with a FlexStation instrument to learn more about using FLEX mode Hard and Soft Parameters SoftMax Pro User s Manual The only settings that must be defined before reading a microplate are the instrument settings found in the Instrument Setup dialog box These set tings tell the instrument what conditions should be in place in order to collect data They include the read mode Endpoint Kinetic Spectrum Well Scan or FLEX the number of wavelengths specific wavelength settings and so on These settings called hard parameters define the conditions under which the data is obtained Once a microplate has been read the instrument settings cannot be changed without deleting the data Soft parameters include the template definition and data reduction settings These parameters can be defined prior to reading a microplate but they are applied only after the plate is read and can be changed at any time In this tutorial you will build a complete protocol based on the settings found in the Default Protocol file which will include both hard and soft parameters You will save the completed protocol file prior to collecting data this protocol file could then be used to generate future data files You should ensure that the instrument that is connected to the com puter is chosen in the Preferences before proceeding in the Edit menu choose Preferences In the dialog box that appears choose your i
621. trum Well Scan or FLEX data in the data display of the Plate or CuvetteSet section Read Mode The method used to read the microplate or cuvette Endpoint Kinetic Spec trum Well Scan or FLEX Note that not all modes are available with all instruments Read Type fluorescence instruments only The type of reading performed in the instrument choose fluorescence time resolved fluorescence or luminescence Readings per Well The number of times settable by the user that readings are taken on a well in fluorescence and time resolved fluorescence modes This setting also deter mines the amount of time that data is collected in luminescence mode Reduced Data Data reduction causes the raw signal values reported by the instrument to be calculated and displayed according to user defined formula settings SoftMax Pro User s Manual Appendix B Glossary of Terms Reduction Limits Sets limits on data to be included in reduction for Kinetic Spectrum Well Scan or FLEX readings Reference A reading of buffer or water taken in the cuvette that is used as Ip to calculate Absorbance or Transmittance of sample read in the cuvette port Relative Fluorescence Units Relative Luminescence Units TER Restore Box Windows if The small box containing both an up and a down arrow at the right of the title bar The Restore box appears only after you have enlarged a window to its maximum size Mouse users can click the Re
622. ts Linear The linear function fits the best straight line to the data The equation for this fit has the form of Y A B X where A is the y intercept of the line and B is the slope A linear fit should be used whenever the values appear to lie on or scattered around a straight line This equation is shown on the screen as y A Bx A linear fit should be used whenever the standard values appear to lie on or are scattered around a straight line Figure Figure 7 43 shows an example of a linear fit a mer Y Standard Curve qi a m m 2 z 30 Concentration y Bx o Std Standards Concentration ws Meanvalue 0 058 Figure 7 43 Graph with Linear Fit SoftMax Pro User s Manual 7 55 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments Semi Log The semi log function fits the best straight line to a set of data for log X plot ted against Y The resulting curve displayed will be a straight line with the X axis drawn in logarithmic scale The equation for a semi log fit is Me B n log49 X and is shown on the screen as y A B log x where A is the y intercept of the line and B is the slope note log in this equa tion is the common or base 10 logarithm we 2 Standard Curve Fit Standard Curve ph x m Cc wm DE E Concentration J A B Logis o Std Standards Concentration ws PTean alue 0 254 Figure 7 44 Graph with Semi Log
623. ts The steepest slope is reported as Vmax Rate If the data plot displays fewer time points data points than the number of time points chosen for Vmax Points then all of the time points will be used to determine the slope of the data Table 8 2 Using Vmax Points Example Time points aens e in the well data plot Number of Points Used for Data Analysis Vmax Points set to 25 25 default Vmax Points set to 5 25 Overlapping Overlapping 5 point line 5 point line segments segments SoftMax Pro will perform a linear regression on as many overlapping 5 point line segments as possible in the analysis window The slope will be determined for each line segment and then the steepest slope will be reported 8 31 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 1 k 8 32 as the reaction rate Thus in well 2 of the example in Table 8 1 above the slope will be determined for line segments consisting of time points 1 to 5 2 to 6 3 to 7 4 to 8 5 to 9 and 6 to 10 and then the slope of the line segment with the steepest slope will be reported as the Vmax Rate The progressive slope calculations are repeated until the last included data point is reached as defined by the End Time or MaxRFU see below The maximum positive or negative slope is reported as Vmax RFU sec Vmax Points can be set from 2 to the total number of points in the curve The num ber of Vmax Points can
624. ts across a Spectrum of wavelengths The default reduced value reported for each well is the wave length of maximum fluorescence or luminescence depending on the read type chosen x Ql ie pe Endpoint Kinetic Spectrum Well Scan Options Fluorescence Wavelengths n Ex 350 Fluorescence RFUS Em Start 350 l Em Stop 750 C Luminescence RLUS Au Time Resolved RFUs Sensitivity Readings 5 PMT Medium Automix Before Off AutoCalibrate On Assay Plate Type 96 Well Standard Figure 5 7 Instrument Settings for a Spectrum Reading All Spectrum readings are made using the scanning monochromator of the SpectraMax Gemini fluorescence readers or FlexStation instruments 5 12 SoftMax Pro User s Manual Chapter 5 Reading Using Fluorescence or Luminescence Instruments Well Scan One or more readings of a single well of a microplate can be taken at a single or multiple wavelength s Values reported are relative fluorescence units RFU or relative luminescence units RLU The default reduced value is Lm1 or Lm2 Lm4 if multiple wavelengths were chosen xi y Ql e Endpoint Kinetic Spectrum Well Scan Read Mode Buttons Options Fluorescence Wavelengths Ex Em Auto Cutoff On 485 538 530 Sensitivity i Readings 6 C Time Resolved RFUs PMT Auto i Automix Before Off AutoCalibrate On Well Scan Editor Pattern Fill Density 3 Fluor
625. tteSet sec tion Notes Menu This menu item changes to show CuvetteSet Plate Notes Graph or Group depending on the type of section that is currently active If no section is active this menu item does not appear Create Summary Edit Summary Create Summary When the Notes section is active choosing Create Sum mary opens a dialog box allowing you to give a title to a new summa ry and then enter a formula to be used for the summary Button Equivalent z in the Notes section Show Formulas Notes Section New Window Edit Summary lIf you have created a summary and need to change it you can do so by first highlighting the summary you wish to edit and then choosing Edit Summary This command is dimmed if you have not yet created a summary or if the summary has not been selected The dialog box then appears showing the name and formula Mouse Equivalent Double clicking the summary performs the same function Button Equivalent F in the Notes section 3 36 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Show Formulas Choosing this command shows the formulas associated with the summaries you have created in the Notes section After choosing Show Formulas the menu name changes to Hide Formulas This com mand is dimmed if you have not created any summaries Section Name Allows you to change the name of the Notes section Mouse Equivalent double cl
626. tting down a run prematurely or if a jam should occur with the tips for example Detailed instructions regarding the procedure to be followed can be found in the FlexStation Operator s Manual SoftMax Pro User s Manual 11 9 Chapter 11 Troubleshooting There is no assay plate mw OFT max Pro e There is no assay plate Please insert a plate before reading You have tried to start a reading without inserting a plate in the microplate drawer Insert the plate and press the button again Communication Problems The instrument icon shown 1 Is the MDC Microplate System instrument turned on in the upper left corner of the 2 If the icon also has the words No port selected through it along SoftMax Pro window has an with an X open the SoftMax Pro Preferences settings and select X through it the proper communications port setting should agree with the physical port to which the cable from the instrument is connected to the computer 3 Quit SoftMax Pro turn off power to both the computer and the MDC Microplate System instrument Check that cable connections between the instrument and computer are secure Turn both machines on again and restart SoftMax Pro If an X still appears over the icon it may be that you are using the wrong type of cable See Communication 1 below for more information 4 If you are using a FlexStation in FLEX mode see Communication 2 below for more information
627. ttings dialog box allowing you to re name a group and or change the sample or group descriptors used as well as to modify the units used with each descriptor Mouse Equivalent double click the name of the Group section Standards to the right of the Group section icon New Window Opens the active section in a separate window Mouse Equivalent double click the FFA icon in the Graph section SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview re P Po fo P P H Window Menu Tile If you have several windows open choosing the Tile command will re evra eps size and arrange all open windows side by side m Cascade If you have several windows open choosing the Cascade command m will resize and layer the open windows so that each title bar will be Lascade visible Arrange Icons Arrange Icons If you have several files open but reduced to icons choosing the Arrange Icons command will move all icons to the bottom of the SoftMax Pro window and put them in order w 1 Untitled Beneath the line will appear the names of all open files within SoftMax Pro The active file will have a checkmark next to it Choosing one of the file names shown here will cause that file to come to the forefront of the screen and will restore the file to its previous size if it had been minimized EPT BETT 1200 Scr 229 H Help Menu Windows Contents Displays the contents of the Help screens allowing
628. type of cable FlexStation Icon Shown with an X If the FlexStation instrument icon shown in the upper left corner of the Soft Max Pro window has an X through it check whether the FlexStation is turned on If itis then check the SoftMax Pro Preferences settings see Setting Preferences below to ensure that the proper communications port modem or printer for the Macintosh COMI or COM2 typically for the PC is selected This setting should agree with the physical port to which the cable from the instrument is connected to the computer If the port setting is correct the instrument is on and communication is still not functioning properly quit SoftMax Pro turn off power to both the computer and the FlexStation and check that the cable connections between the instrument and computer are secure Turn both machines on again and restart SoftMax Pro If an X still appears over the icon change the setting for the Serial Comm Speed found in the Preferences dialog box choose Edit gt Preferences If the setting is cur rently 9600 change it to 57600 or vice versa Macintosh Computers Having a Single Serial Port Some Macintosh computers have only a single 8 pin DIN serial port which serves as both the printer and modem port Please check to see if your model is listed as compatible hardware on the SoftMax Pro for Macintosh Version System Requirements and Compatible Hardware sheet If your computer is not listed the per
629. type of group is selected in the Group drop down list The following illustration shows how the tool bar will appear under various circumstances Cla sp L lear Edit Sample eee n zu v lear When Plate or Template Blank is selected Clear Group Unknowns cv t ba A group is selected that does not contain sample descriptor information such as Unknowns column type GJ smpe Sea Group Standards Y Concentration bo zug ml A group is selected for which a concentration has been defined such as Standards column type Group Unknow ution Y spen TS Dilution Factor O A group is selected for which a dilution factor is defined such as UnknownsDilution column type Figure 5 18 The Appearance of the Template Editor Tool Bar under Different Circumstances 5 21 Chapter 5 Reading Using Fluorescence or Luminescence Instruments 5 22 The tool bar is divided into three areas the Group area the Sample area and the Assign area The Group Area The Group area contains the Group drop down list which allows you to assign wells to existing groups create new groups or modify using the Edit button existing groups In addition it contains the Clear button which allows you to clear well assignments in the template Group Drop Down List This drop down list consists of two areas divided by a horizontal gray line The two items below the gray line Blank and New are always av
630. u might forget your pass word write it down and store it in a safe place for future reference SoftMax Pro User s Manual 9 31 Chapter 9 File Management and Printing Plate 4 a Setup E Template Eg Display p Plate 1 Hes ieee eee iade 5 This file is password protected You must enter the correct password before you can save 1885 5 2085 5 2285 5 2485 modifications 2250 5 2465 5 2605 5 2585 2585 2 2885 2 3185 25 5485 S183 5 2483 5 2785 5 4085 U Suspend Password 3785 5 4085 5 4405 5 4085 A E D E F G H 4485 5 4585 5 5385 5 5885 Wavelength Combination Lrn Cancel Author Name Author Figure 9 18 Password Required Dialog Box When this dialog box appears you must enter the password in order to con tinue with the action you have chosen Note the Author s name shown in the lower left corner of the Plate section This Author s name and the password are tied together in an electronic signature Once an Author s name has been entered for a file it can not be changed unless password protection is removed If you do not remove password protection prior to trying to change the file every attempt to change the file will cause this dialog box to appear Enabling the Suspend Password option click the box to check it will stop this repeat prompting 9 32 SoftMax Pro User s Manual Chapter 10 Tutorials Introd dc OB aeo Gusset edu cde SLM sedet Sera se teile d pig 10 3
631. uch formulas for Kinetic and Spectrum readings Table 7 3 Kinetic and Spectrum Reduction Formula Examples Vmaxcorr combinedplot Reports Vmax correlation coefficient for plots in all 96 wells Ivmaxpoints readinterval Vmax Delta combinedplot Will report the Vmax Rate of the delta between each time point Ivmaxpoints readinterval Spectrum or Kinetic Nthitem Lm1 X Will report the optical density at item X in the list of readings For example if you have a Kinetic run with 20 time points and X is 10 it will report the OD of the 10th time point Similarly if you have a Spectrum scan with 20 measurements and X is 10 it will report the OD of the 10th wavelength measured For a full discussion of custom formulas see the Formula Reference Guide When you finish your entry in the Calculation dialog box and click OK the formula you have entered then appears to the right of the word Custom and becomes the default selection for the Custom option You may subse quently edit this formula or enter a new one by clicking the Formula button again Recalculation Options By default SoftMax Pro performs continuous recalculation of the data when you read plates create or change formulas and so on At certain times it would be useful to disable the recalculation feature so that you can make alterations to the experiment without waiting for individual recalculations to be completed Two commands are available in the Edit menu tha
632. uded in data reduction but do not affect data collection If you alter a limit to show less data you can always display the hidden data again by changing the limit MaxRFU MaxRLU The limit for the maximum value you wish to report Any values from the reading that are above this limit will not be shown and will be excluded from data reduction The default is 20 000 for RFU RLU The MaxRFU RLU reduction parameter can be used to exclude the non lin ear portion of the reaction from data analysis This type of data reduction is most useful in reactions where the initial portion of the data is linear You might also adjust the End Time setting to remove a non linear portion after a certain time point in the reaction Using MaxRFU RLU allows the use of the maximum number of linear points to calculate the slope of the line and thus to determine the rate for each well 6 21 Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments 8 22 mi Plate 1 Well Graph Reduced iB xl E z oO 50 100 150 200 250 300 Time secs Wimax Points 34 Vell C4 n DS A EB Wimax Per Second 76 285 85 948 987 440 R 2 0 924 0 538 0 356 BSE at 50 100 150 200 250 300 Time secs Wimax Points 34 Meli o C4 aps A EB Ymax Per Second 50 082 55 850 65 557 R 2 0 958 0 870 0 983 Figure 8 14 Different Reductions of the Same Data Using Different MaxRFU Points Both of the Well Graphs sho
633. ue up to 999 seconds in the box to the right Vmax UVmax and ThermoMax Instruments Endpoint Kinetic Automix amp Blanking 1 ae gt E Automix amp Blanking p Automix pesAutomix _ Before First Read LL Before First Read Between Reads Figure 4 14 Automix Settings Box for Vmax UVmax and ThermoMax Automix read intervals for the Vmax UVmax and ThermoMax are not user definable The Emax does not have Automix capability Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments k Before First Read This setting can be used with either Endpoint or Kinetic readings Click the checkbox next to Before First Read to shake the microplate before the first reading automatically done for five seconds Whether you read at a single wavelength or dual wavelengths enabling Automix for Before First Read will shake the plate for five seconds before the first wavelength reading only Between Reads This option is applicable only to Kinetic readings and will be displayed only when Kinetic mode is selected To enable automatic mixing of the microplate between readings during a Kinetic assay click the checkbox next to this set ting For readings at a single wavelength Between Reads will shake the plate for three seconds prior to each reading at that wavelength For dual wavelength readings enabling Between Reads will shake the plate for three seconds before each reading at the first wavelength only R
634. uid time points P Transfer scheduled after read has stopped Check total read time Alerts in SoftMax Pro The Alerts that follow are presented alphabetically by first line of the alert message Low or Sat Messages Plate Section Data Sat 1873 8 1972 8 2072 8 4312 8 2772 8 3172 8 3472 8 3772 8 1972 3 2072 3 2172 8 4512 8 a472 3472 8 3772 8 4072 5 e E cm cu alras asrae we a2 sow patens eren err ees owe mz Wells Pd reportin 4572 2 Boat 2972 8 3472 2 Heat 4072 2 4372 2 4772 2 Boat po Edi 6172 8 P 8 pa 8 Low Sa 3172 8 HLow 3772 5 4372 8 4772 8 5172 8 Haar ATR 0712 8 7312 8 mr A and Sat 3112 8 1072 8 4772 8 5172 8 serak asa irre 7372 8 9072 8 errors HLowr 4372 8 5172 8 5672 2 6172 8 E Erz 9072 2 9272 8 10673 4112 8 5672 8 6172 8 6772 8 5072 8 8872 8 9772 5 11773 Lows NOIA These reports may appear in the Plate section of fluorescent reading instruments during Endpoint mode The error Sat appears when the well is saturated i e too much signal Try lowering the PMT setting and re read the data If the well is still saturated after the PMT is set to Low try diluting the sample The error Low appears if the flash lamp missed the flash for that well Reread the affected well s SoftMax Pro User s Manual 5 Chapter 11 Troubleshooting A plate descriptor used by this file is for another instrument W SOFT
635. ula 10 65 FTINGANCDOIM i cesis PSdS 4d Fundus eane nd dede as 10 67 Chapter 11 Troubleshooting 0c ccc ecw ec cece eere 11 1 n EROC OC HOT oa do Eure a onset eens E RENE 11 3 Message Boxes and Alerts 0 0 eee eee 11 3 Alerts in the Instrument Settings 00 11 3 Alerts in SOM Max PEO s ie qu eod de t Er doe d dodi 11 4 Itt OF Message DOXCS se ed oio beaks det hia ey eae eae rs 11 8 Communication Problems 0 00 c cece eee eee 11 10 Display TrODICMIS n22smne e erdeeed here eoneas E S 11 14 Printing Problems isis erue 13 ERES Sa HE gud Pierii 11 14 General Problems PRETERITO o o 11 15 Chapter 12 SoftMax Pro Remote Command Language 12 1 SNOCHCHOB cuu eed soos dbe d PE ERR qoa V Aa ane seen S acu nae 12 3 Notes On Queued Processing sleleseseeeesse 12 3 Send Receive Remote Commands 00 eee ee eee eee 12 4 Summarizing Remote Commanding 005 12 7 PLOSTAMMEr INOS e 2405 tice rear quias d AIR CA eee RRA 12 7 Molecular Devices Position 0 00 cee eee eee 12 8 SoftMax Pro Commands i2 22e eg ose chan onbres eria dendi arde 12 9 Visual Basic Excel Macro Example lesse 12 14 MFC C Interface to SoftMax Pro Remote Commands that Return ValueSeusosim eser etre mbar i ace 12 15 Appendix A SoftMax Pro Version Specifications A 1 D EEOC DGHOTI 2o 52 soon 2p toph nico t
636. ult reduced values calculated for Kinetic data are Vmax mOD min Time to Vmax seconds Onset Time seconds Time at Minimum Time at Maximum Time at 1 2 Maximum Slope and Area Under Curve ri Endpoint Spectrum Options Wavelengths 450 Run Time 5 00 Interval Number of Reads 26 Time 5 00 Interval 0 12 26 Minimum Interval 0 09 Automix amp Blanking Before Off Between Off Pre Read Plate Off AutoCalibrate On Strips Read entire plate AutoRead Minimum Interval 0 09 Nonna enna ee R AREE RR ER ER ERR ERR RERE RANA RR RR RR ERES ER ER ES ES ESOS EASA EEED ESOL ERSESES EROS ESOS ER hern hn nn nnn Figure 4 5 Instrument Settings for a Kinetic Reading Timing When Kinetic mode is chosen the Timing parameter is active allowing you to enter the total Kinetic run time and the time interval between readings A default value will be shown for the run time to change this value click in the box Up and down arrows will appear enabling you to increase or decrease the value shown Highlight the hours minutes or seconds shown and then click the up or down arrow until the desired value is reached You can also select an existing value and enter a new value using the keyboard After entering a total run time and an interval between readings the total number of readings that will result is shown below the run time The mini mum read interval is determined by which instrume
637. um Lrmi Example Raw Bata Example Options Plot j 0 320 v Number VS Threshold Bede d i Time j Ranged ISp ay j j Grayscale Choices TER default Plat Show E with reduced number Cancel Figure 7 2 Plate Section Endpoint Kinetic and Spectrum Display Dialog Boxes SoftMax Pro User s Manual 7 7 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments t am Cuvette Display xc Be rs Redpiced default ju Endpoint ex Example E Reduced Dey T NENNEN Choices Show Choices for vith reduced number Cuvette Layout Cancel CuvetteSet Section W Cuvette Display Raw Data Plot xd Kineti OD vs Time Raw OD EHE default j Example DTE pee mE Show With reduced number Cancel mg Cuvette Display ax Raw Data raw Reduced Plot S Spectrum OD vs Wavelength Faw oP default j Example X Three Samples Per Fw Show With reduced number Cancel Figure 7 3 CuvetteSet Section Endpoint Kinetic and Spectrum Display Dialog Boxes 7 8 SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments SoftMax Pro User s Manual Data in the Plate section is displayed in a grid array microplate format With all plates other than 384 well plates the display is always shown as a stan dard microplate with well A1 in the top left corner If you are readin
638. um OD and decrease the number of Vmax Points used to deter mine the slope of the line SoftMax Pro User s Manual 7 29 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 30 MinOD The limit for the minimum value you wish to report Any values from the reading that are under this limit will not be shown and will be excluded from data reduction The default is 0 OD To display negative Kinetics the value should be set below 0 zero Lag Time Specifies how many initial data points are excluded from the calculation of Vmax Rate Lag Time truncates the data used in the calculation It does not prevent data from being collected The Kinetic plots will not display the data collected prior to the set Lag Time The value for Lag Time can be changed after the plate is read The default is 0 Bj Plateiti Well Graph Reduced ILm1 Time secs 2 Ymax 52474 69 137 R 2 1 000 0 977 220 240 260 Time secs Figure 7 22 Different Lag Time Settings In the figure on the top left well C5 has a lag time the data is not linear until approximately 80 seconds after the start of data collection Thus the linear curve fit is not good and the slope of the line does not reflect the maximal rate In the figure on the bottom right a lag time of 80 seconds has been set and the linear curve fit for both wells is good allowing the slope of the lines to reflect the maximal rate in each well Alternativel
639. und amp Tip Columns Mone Figure 10 51 Instrument Settings Completed Click to accept the settings and close the dialog box SoftMax Pro User s Manual 10 47 Chapter 10 Tutorials Step 3 Define the Template A Click the Template button in the Plate section tool bar This will open the Template Editor showing all wells empty Drag the mouse or shift click to select the first column of wells A1 through H1 The Group pull down menu on the Template Editor shows Blank as a default and the only other choice is New Choose New to create a new group In the dialog box that appears enter Data in the highlighted text box to name the new group Then click the drop down menu in the Column Format section and choose Unknowns dilution from the list Doing this will cause a checkmark to appear in the box next to Sample Descriptor and automatically enter the word Dilution Factor in the text box below that The dialog box should appear as shown in Figure 10 52 W Group Settings x Name rv Sample Descriptor pretacoudssansere coaveusssusvetsceuasens rete E PATE Dilution Factor Units vj Figure 10 52 Group Settings Dialog Box for Data Group Click to close the dialog box The template should now look like the illustration below m Experiment 1 Plate 1 E X ET Date F Eme Datani series Data Y DiuienFacto n SF qw 6 m 5 x Data
640. ure of the program and places a checkmark to the left of the command in the menu to indicate that it has been selected While this function is enabled no recalculation will occur regardless of what you may change add or delete from the experiment This is especially useful when creating or changing col umn formulas within Group sections for example as it can speed up the pro cess greatly When recalculation has been suspended you may wish to see the results of the changes you have made but may still not wish to enable continuous recal culation Choosing Recalculate Now 3 CTRL from the Edit menu will cause SoftMax Pro to examine all elements of the experiment and perform complete recalculation of the data Choosing this command does not enable continuous recalculation but simply causes the program to perform recalcula tion once To return to continuous recalculation choose Suspend Recalculation again The checkmark next to the command will disappear indicating that the function is no longer in use AOINA Recalculate Now 3 CTRL can be used to see intermediate results during long Kinetic readings At any time you can choose Suspend Recalculation to delay calculations until a later time Displaying Data in Group Graph and Notes Sections 8 38 Group Sections The manner in which data is displayed in a Group section depends on the type of reading that was performed Endpoint Kinetic Spectrum Well Scan or FLEX
641. ure 3 14 Graph Section To create a graph section choose New Graph from the Experiment menu The Plots dialog box opens automatically and a default plot called Plot 1 is selected The plot will default to the first group available in the experiment Once a graph has been created new plots can be added and deleted the axes can be customized and the size displayed area of the graph can be changed Grid lines can be enabled or disabled default is enabled Graphs may be dis played as bar graphs or scatter plots The Graph section is divided into three areas e The tool bar e The body of the Graph section e Plots information below the graph 3 21 Chapter 3 SoftMax Pro Overview l F You may create more than one Graph section within an experiment Plots in the Graph section must be defined from groups within the same experiment section as the graph with one exception if you duplicate an experiment that contains data in the Plate section and has a plot defined in a Graph section that plot will remain in the duplicated Graph section and new plots may be added The Graph menu contains the following items shown with their tool bar or mouse equivalents These menu items and buttons are active only when a Graph section is active Table 3 5 Graph Section Menu Commands and Tool Bar Buttons Menu Command m New Window in the Opens the Graph section in 7 double click Graph menu a new window Name of Graph sec Opens the S
642. ure 7 17 Figure 7 18 Figure 7 19 Figure 7 20 Figure 7 21 Figure 7 22 Figure 7 23 Figure 7 24 Figure 7 25 Figure 7 26 Figure 7 27 Figure 7 28 Figure 7 29 Figure 7 30 Figure 7 31 New Password Dialog BOX ned v aet Ee e wed apt om dub uatwi sanean 6 20 Users Property Page Dialog BOX 25a e mr Ro hee d hn aed 6 21 Global Opis Pane siibet ed dud a d adii dd dd DE 9 ddr see 6 22 Report LODE y Dae e DOX couette dra arp dad drag and ed renew diode 6 23 Export File Save As Dialog DOXauaus muta dams odes p opes ud dee oru d a eds 6 24 Sample Export File Opened with Notepad ssseesee eese 6 24 Andit DD DOX coq 40 92 2 9 9 uice ee EH quie ire iater edd qn cade ea dua 6 26 License Property Page DOX au wa cnet ea Eur she is doses iod d ado ACE RUP RE ae bris 6 27 New License Record Dialog Box 1 0 oes 6 27 Security Menu in SoftMax Pro Enterprise Edition 000 005 6 28 User Information Dialog BOX sca eactonuca mui dca qi QUE deg Gon ess quld sceau 6 29 Electronic Statement Setup Dialog Box 0 0 0 ce eee ee 6 30 Statement Section in SoftMax Pro Enterprise Edition 0 6 30 Statements Menu in SoftMax Pro Enterprise Edition 0 6 31 SIETEDIQte rHent Palo DOX coss uae acte ure diri quique ars atre eant oa din mscedid ei dec 6 32 Sioned 5Edtenient DeC HUON s serrr srr prier p ey darts eris mda m PR RR Vp A RE deles 6 32 Relationship of Secti
643. used for han dling OD and T calculations for Pre read plate blanking and Path Check because OD calculations are performed on a linear scale whereas T calculations are performed on a logarithmic scale How ever other calculations are not calculated differently by SoftMax Pro for absorbance and T modes Therefore switching from 76 T to Absor bance or vice versa is not recommended Additionally Molecular Devices recommends that 7o T be used only for raw OD raw OD with Pre read plate blank subtraction or PathCheck corrected raw OD readings Use caution when using T on reduced numbers or any readings that apply other calculations since the data may not accurately reflect proper calcula tions If you switch from one mode to the other note that settings for the two modes are not linked settings chosen for one mode are not reflected if you switch to the other mode For example if data from a Spectrum scan is displayed with a MinOD of 0 and a MaxOD of 1 absorbance and you then switch to Transmittance and change the limits to 0 and 5076 respectively switching back to absorbance will not change the limits set previously the Min and MaxOD limits will still be 0 and 1 SoftMax Pro User s Manual 7 37 Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments 38 Custom Reduction Formulas If the predefined reduction formulas do not meet your needs you can create one or more different reduction formulas
644. uuneueuuuneneumunen Show Iv With reduced number Cancel Figure 10 59 Display Dialog Box The default display for FLEX readings is raw RFU This tutorial example shows a reduced number so make sure the checkbox under Show is checked If you decided later that you would like to view reduced data you can change the display options after the reading since this does not affect the actual data only how it appears on the screen and printout Click to close the dialog box 10 52 SoftMax Pro User s Manual Chapter 10 Tutorials Step 6 Save the File Choose Save from the File menu jj Tutorial Date Modified Name mtitted Format 5oftMAX PRO Document 5 0ft MAX PRO Protocol Figure 10 60 Save As Dialog Box Macintosh Save this document as P A3 x Save In E Protocols amp j E Fal File name Tutorial pr save as type Fro Protocol Files ppr Cancel Figure 10 61 Save As Dialog Box Windows On the Macintosh choose SoftMax Pro Protocol from the drop down menu next to Format In Windows click in the box under Save File as Type and choose Pro Protocol Files ppr Enter the name for the file in the text box highlighted over Untitled in this example type My Tutorial Then click Save to save a copy of the protocol file on the hard disk The title bar of the window will still show Untitled rather than My
645. v series le Group Data w pm a Clear Dilution Factor 0 01 EET 23 3 4 5 12 Figure 10 54 Template showing First Samples Data Concentrations A total of eight samples will be assigned to columns in this template Con tinue to highlight subsequent columns of wells and enter the following dilu tion factors Column A3 through H4 0 05 Column A5 through H5 0 5 Column A6 through H6 1 Column A7 through H7 5 Column A8 through H9 10 Column A10 through H10 100 The template should look like the one in Figure 10 55 when you are finished entering the samples 10 49 Chapter 10 Tutorials m Experiment i Platez1 j xj E EA o EE Dataos baee TV ere Betas E Pah Clear Dilution Factord D 1 z 3 4 2 5 T a g 10 Figure 10 55 Template Filled with Samples D The template is now complete Click the button to accept the entries and close the Template Editor The outlines of the groups you have defined in the Template Editor will now be shown in the Plate section v Ca aer a v Max units per sec Plate 1 1 2 3 4 5 B T B J 10 i FLEX Time 200 secs Interval 2 secs Reads 101 Fluorescence i Bottom read Ex Em Cutaff Lind 485 525 515 Auto Cutott I coi m 7 0 m I Wavelength Combination Lrn1 Automix Off Calibrate On PMT High Readsivell 3 Lag Time 0 End Time 200 RFU Min C RFU Max 20000 max Pts 107401 Plate Last Read Mo Da
646. ve as command Append to File when saving as Text file only If you are saving data to an ASCII text file you may choose to save all data to a single file for all plates and or cuvettes by choosing Append to File This option is dimmed not available if you are saving to a SoftMax Pro file File Prefix The information entered into the File Prefix field is automatically given to new files that are saved automatically The default prefix is Data Accepting this default saves files starting with the name Data followed by a sequential number beginning with 1 For example the first microplate read would be saved under the file name Data 1 Preferences File Prefix Field m Autosagwe EI Bu ue rte ee ice B Wi Append Date File Prefix Fi Append Time i SOFTmax PRO File 3 Text File Figure 2 15 File Prefix Field Windows You can change this default prefix to any other prefix up to 15 characters To change the prefix click within the File Prefix field highlight the existing infor mation and type over the word to enter a new prefix If the File Prefix field is left blank the date of the run will be used as the file prefix Append Date Enabling the checkbox for Append Date will cause the date to be included in the automatic file name for the saved data file For example using the file pre SoftMax Pro User s Manual 2 21 Chapter 2 Installation 1 k fix Data the fi
647. velength combination reduction This setting uses all visible timepoints in the reduction window Slope is the same as Vmax Rate when Vmax Rate is set to the same number of points as the run i e the default Slope is not the same as Vmax Rate if you have modified the Vmax Points Area Under Curve This reduction estimates the area under the curve as defined by the data plots in wells within the reduction limits The data plots are treated as a series of trapezoids with vertices at successive data points and at the X axis coordi nates of the data points The areas defined by each of the trapezoids are then computed and summed Absolute Values Choosing this option shows the raw data without any smoothing or other changes in the way it is graphed SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments Spectrum Spectrum Limits The Limits settings for the display of Spectrum data are MaxRFU RLU Min RFU RLU Starting Wavelength in RFU RLU and Ending Wavelength in RFU RLU The display of values is shown relative to the first point measured for each well Limits define the data that will be viewed and included in data reduction but do not affect data collection If you alter a limit to show less data you can always display the hidden data again by changing the limit MinRFU MinRLU The limit for the minimum value you wish to report Any values from the readi
648. verage of the blank values obtained from the Blank group is subtracted from all wells in the plate Group Associated Blanks are created by placing blank wells within a group in the template The values of group blanks are averaged and subtracted from the values of the wells within that group only Bottom Read FlexStation only Available in the Instrument Settings this selection causes the FlexStation to read up through the bottom of the microplate rather than down from the top Checkbox A small square box that appears in a dialog box and that can be selected or cleared When the checkbox is selected an X appears in the box A checkbox represents an option that you can turn on or off Clear Within the Template Editor Clear removes information that was designated previously for selected wells By highlighting wells and then choosing Clear any information regarding those wells will be erased the wells will return to their default status of empty Note that if you clear the entire template the groups themselves will not be deleted and are still selectable Keeping the groups allows you to reassign wells without having to recreate groups Click You click an object on the screen by pressing and quickly releasing the mouse button while the pointer is on that object When you click once on an object such as an icon or file name it becomes highlighted which means it is selected Whatever action you choose next will affect a select
649. w Column Edit Formula Plots File gt Save As Edit gt Undo Edit gt Cut Edit gt Paste Edit gt Clear Edit gt Delete Section Edit gt Duplicate Section Graph Options X Axis Y Axis All Section Set tings Edit New Experiment Edit gt New Section Edit Text Style Control gt Instrument Setup Notes all items Plate all items Group all items Graph all items Cuvette all items Only if data exists in the section 9 30 SoftMax Pro User s Manual Chapter 9 File Management and Printing Setting and Changing a Password Choosing Set Password from the File menu causes the Password dialog to appear If the active document is already password protected the menu com mand changes to Change Password Passwords can be from 1 to 8 charac ters long and can include all characters available from the keyboard You must also enter an Author Name that will be saved and printed with the data Set Passvoard 5hielding mu set Password 5Shielding Author Name and m Author Name and Old Password pase e New Password i i New Password Retype New Password j Remove Password L Shield Protocol File O Shield Protocol File Protocol Password Protocol Password tetype Protocol Password ietype Protocol Password Cancel Figure 9 17 Set Password Sheilding Dialog Box Left no password set previously right with previously set password This dialog box appears diffe
650. w in the group pull down menu and the Group sec tion will still exist Group Settings Name Sample Descriptor AE E E E E E E Concentration Junita ml gt Column Format Figure 4 20 Group Settings Dialog Box Using Default Settings 4 24 SoftMax Pro User s Manual Chapter 4 Reading Microplates and Cuvettes Using Absorbance Instruments SoftMax Pro User s Manual Name Use either the default name Group X where X is an integer that is initially set to 1 and increments i e 1 2 3 etc as other groups are created or enter a custom name up to 32 characters in length In the Sample Descriptor area of the Group Settings dialog box you can enable the application of the sample descriptor if appropriate by clicking the checkbox You may also choose the units used for the sample descriptor by selecting an option from the Units drop down list or by typing the units you wish to use into the list box The Sample Descriptor is automatically enabled when either the Standards or Unknowns Dilution column format see below is selected The descriptor defaults to Concentration with the Standards column format and to Dilution with the Unknowns Dilution column format The Sample Descriptor may also be used for time or fraction number information No sample descriptor is assigned automatically for the Basic or Unknowns column format You may assign a sample descriptor to these column formats manu
651. w or change information that you have entered If you purchased the Drug Discovery version of SoftMax Pro a default proto col will be installed automatically If you purchased either the Life Sciences or Enterprise Administrator version of SoftMax Pro however you will be asked to choose whether the default protocol should be absorbance or fluorescence If an instrument is connected to your computer SoftMax Pro will automati cally detect the appropriate instrument setting If no instrument is connected choosing absorbance will assign the SpectraMax 190 as the default choosing fluorescence will assign the SpectraMax Gemini XS You may change either instrument preference later During installation an Assays folder is created which contains protocol files for all instrument types regardless of the choice made above Some of these protocols will not be appropriate for your par ticular instrument they might have reductions that are appropriate only for absorbance or fluorescence instruments for example Nev ertheless all protocols can be opened run and or customized to be appropriate for any MDC Microplate System instrument Click Next to start the actual installation of SoftMax Pro files When installation is complete the Finished screen will appear Click Close to end the installer setup program On the Macintosh remove the CD by dragging its icon to the Trash On the PC close the window for the CD and My
652. well in a plate or one cuvette When template information is exported the information for well cuvette A1 is written into the first line of the file information for well cuvette A2 is writ ten to the second line of the file and so on until a line has been created in the export file for each well in a plate or all cuvettes If no template information exists for a well in a plate or for a cuvette that line is left blank The ASCII file terminates with the string End The order for importing template information must match the export order Similarly blank lines in the file are assigned to a well or cuvette providing a well cuvette with no template setting Table 9 3 describes the information that is included in each column of the ASCII template file Table 9 3 ASCII Template File Columns and Descriptions t pee name Assigned in Assigned in the Group Settings dialog box If this field is missing the rest Group Settings dialog box If this field is missing the rest oe the line is ignored Any text string can be entered in this field s s for Assigned in the Group Settings dialog box Five text strings are supported in SoftMax Pro 4 1 Empty Basic Standards Unknowns and Unknowne Dilu tion Blank groups have an Empty column format If this field is empty the Basic column format is assigned Sample name The sample name is set in the Sample field of the Template Editor dia log box If this field is left blank no sa
653. wn above display different reductions of the same Kinetic data In the figure on the top the MaxRFU is set to 20 000 for the dis play of wells C4 D6 and E6 In the figure on the bottom the MaxRFU has been reset to 10 000 Alternatively you could leave the Maximum RFU and change the number of Vmax Points used to determine the slope of the line MinRFU MinRLU The limit for the minimum value you wish to report Any values from the reading that are under this limit will not be shown and will be excluded from data reduction The default is 0 RFU RLU To display negative Kinetics the value should be set below 0 zero SoftMax Pro User s Manual Chapter 8 Displaying and Analyzing Data Using Fluorescence or Luminescence Instruments SoftMax Pro User s Manual Lag Time Specifies how many initial data points are excluded from the calculation of Vmax Rate Lag Time truncates the data used in the calculation It does not prevent data from being collected The Kinetic plots will not display the data collected prior to the set Lag Time The value for Lag Time can be changed after the plate is read The default is 0 I Plateit Well Graph Reduced IL m1 Time secs Vmax Points 17 Well o A1 2 Ymax 52 174 69 137 R 2 1 000 0 977 220 240 260 280 Time secs Figure 8 15 Different Lag Time Settings In the figure on the top left well C5 has a lag time the data is not linear until approximately 80 seconds af
654. x Pro menu bar is shown when a changes to reflect the type of section that is active If a Plate section is active for example this menu item will show Plate If an experiment or multiple sections are selected this menu item will not be shown New Plate Cuvette Notes or Graph sections can be created whenever you need them and more than one of each type of section can exist in a single Soft Max Pro experiment Group sections are created automatically when you define new groups in the Template Editor or may be created with the Dupli cate command A section can be deleted if it is no longer needed The Delete command in the Edit menu changes depending upon which section is active For example if the section titled Plate 1 is active the Edit menu will show Delete Plate 1 Using the Macintosh you can also delete the active section by pressing 3 within Windows you can press CTRL Take care when deleting sections that they do not contain information you wish to save You may also delete SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview multiple sections simultaneously by making them active the Delete com mand in the Edit menu will change to show Delete Selection Deleting a Group section automatically deletes references to that group from the Template See either Chapter 4 or 5 choose the chap ter that is appropriate for the instrument you are using for more information Section tool bars contain
655. xamples 3 3 Chapter 3 SoftMax Pro Overview F After a few seconds the title screen will be replaced by an untitled SoftMax Pro window The window for the Macintosh should now look similar to the one in Figure 3 2 The window for the PC should look similar to the one in Figure 3 3 The SoftMax Pro screen contains these major elements e A menu and status bar located at the top of the screen e An untitled window showing an experiment containing various sections depending upon the configuration of the default protocol used and the type of instrument chosen in the Preferences or connected to the computer The menu and status bars are always visible while you are using SoftMax Pro no matter where you are within the program Menu Bar Title Bar Active Print Tool Bar Rn EE File Edit View ee ae Control Assays Help Sots Rar eer Dmm I By oe m Untitled p lt i B 1 OX Experiment T L5 Experiment 1 Section Tool Bar v E netes ET Notes Section Arrow LL Plate 1 Open Close Triangle Endpoint Lmi 450 Eellbests On alibr ate gt Un Plate Section NNUS Vertical Mo Data Scroll Bar Wavelength Combination Lrni Data Mode Absorbance Figure 3 2 SoftMax Pro Screen and Window for Macintosh 3 4 SoftMax Pro User s Manual Chapter 3 SoftMax Pro Overview Status Bar Experiment Section Tool Bar Notes Section Plate Section Data Display Open Close Triangle Indicator
656. xperiments are created beneath the other experiments Duplicating a CuvetteSet section creates a copy of the previous CuvetteSet section including the last reference that was read 9 27 Chapter 9 File Management and Printing New Table 9 4 New vs Duplicate within an Experiment Notes Section Plate Section Cuvette Set Sec tion Graph Section Group Section 9 28 Group section tables can also be duplicated When a Group table is dupli cated its name is added to the Template Editor s list of group names but the group is not assigned to any wells Graph sections that are duplicated include all settings for plots fit etc Notes sections are duplicated exactly including any summaries and all text New sections and new experiments can be created by selecting New from the Section or Experiment menu If an existing section or experiment is active when this command is chosen all of the information in the section or experi ment except data is copied to the newly created section or experiment The name of the new section or experiment is the same as that of the original with the addition of an incrementing number e g OldName 2 A new section is created immediately below the active section A new experiment is created immediately below the active experiment Creating a new CuvetteSet section creates a copy of the previous CuvetteSet section including the template if any but this section contains no
657. y you could leave the Lag Time and decrease the number of Vmax Points used to determine the slope of the line SoftMax Pro User s Manual Chapter 7 Displaying and Analyzing Data Using Absorbance Instruments End Time This setting specifies the time at which you wish to stop showing data in the display Any values occurring after this limit will not be reported in the dis play The default setting is the total assay time The value for end time may not be greater than the total assay time Absolute Values This setting causes the plots shown in the data display to be drawn at their absolute OD as opposed to the default with the box unchecked which offsets the first point to the 0 0 coordinate Enabling Absolute Values may cause some data points that do not fall within the default limits for the plots to dis appear see Figure 7 23 vMax Points 1 rData Mode Lag Time amp absorbance End T ime j Q STransmittance w Absolute Values v Fa Fatet E Cx Kinetic Time 00 Interval O25 13 Lag Time 0 00 End Time 3 00 Ir c m m vc oO um F OD rax 0 5 Ymax Pts 13 1z Wavelength Combination Lm1 Data Mode Absorbance Flate Blank Used Flate Last Fead 10 51 AM 77 16799 Figure 7 23 Kinetic Reduction with Absolute Values Enabled Note that the limits in this example MinOD 0 MaxOD 0 5 do not allow all of the data to be vis
658. y new cuvettes created in the CuvetteSet section after reading a second reference will use that new reference reading When a reference is read the system assumes that it will be used with all empty samples Therefore the date and time of the reference reading are dis played with each empty sample including the destination sample currently in the CuvetteSet section If you wish to use a different reference reading with one or more cuvettes in a section click or shift click the cuvette s and then click the Ref button A dialog will appear asking if you wish to replace the reference reading for the cuvette s Choosing will enable a new refer ence reading to be used for those cuvette s The reference read is automatically applied to the normal read The reference value is not displayed in SoftMax Pro If you wish to see the reference value you can choose one of the following procedures Procedure 1 1 2 1 2 3 4 Create a cuvette in the CuvetteSet section and Ref on air empty cuvette port Read a cuvette containing the blanking solution If you wish to have both reference and read values available do the following Create a cuvette in the CuvetteSet section and Ref on air empty cuvette port Define a template blank in the CuvetteSet section Place buffer in the cuvette and Read Read the remaining samples The optical density of the cuvette designated as the template blank will be subt
659. y or to begin the first reading of a Kinetic plate faster However you should allow the instrument to perform autocalibration of at least one plate prior to disabling this function calibration settings for the last plate which included an autocalibration are maintained in the instrument memory NVRAM until another autocalibration is per formed Assay Plate Type This setting determines the display of wells in the microplate it should be set to match the type and number of wells in the actual microplate that will be read Select an assay plate type 96 well plates 96 Vell Standard 384 well plates 384 Yell Standard 48 well plates 48 Well Costar 24 well plates 24 Vell Costar 12 well plates 12 Well Costar 6 well plates Figure 5 15 Plate Type Setting Clicking in the box allows you to choose the number of wells and type of microplate you will be reading Changing the Plate Type within a Plate section causes well assign ments stored in the previous template to be discarded The groups created previously remain however so you can select new wells and simply apply existing groups to these wells Defining New Plate Types The Plate Type list includes entries for microplates with standard dimensions and additional entries for plates that work best with custom settings For both 96 and 384 well plates the Standard plate should be used unless a custom setting is shown for the microplate you are using Wells to
660. ype 5 13 Reader pop up menu 2 18 Recalculate Now command 3 29 7 40 8 37 Recalculation options 7 40 8 37 Reduced Number display 7 14 8 8 Reduction 1 3 7 4 7 23 7 26 8 4 8 19 command 3 33 3 35 custom formula 7 38 8 35 defaults 7 27 7 28 7 33 7 36 8 21 8 26 6 29 8 30 kinetic 7 33 8 26 options 7 4 8 4 parameters 7 3 8 3 spectrum 7 36 8 29 Reduction Display Options 8 34 Ref Cuvette Set 4 33 Ref button 3 8 Reference problems 11 15 reading 4 41 Registration dialog box 2 13 Replicates 4 30 5 27 Report 9 12 customizing 9 12 default setting 10 34 10 67 printing 9 11 9 12 10 34 10 67 including excluding sections 9 12 Report Enterprise Administrator 6 23 Report Page 6 12 RFU RLU values absolute 7 18 7 31 7 32 6 24 8 25 8 28 8 34 ROBOmax 9 10 SoftMax Pro User s Manual S Sample descriptor 4 25 5 23 Save command 3 26 9 5 9 8 10 21 10 53 Save As command 3 26 9 5 9 8 Saving files manually 9 8 Scroll B 13 Scroll bar 3 11 Section 3 9 active 3 10 3 11 3 24 3 27 3 28 3 30 3 34 B 1 changing order 9 13 creating 3 10 CuvetteSet 3 17 deleting 3 9 3 10 Graph 3 10 3 21 7 47 7 54 curve fit 7 53 7 54 8 51 options 7 65 8 62 plots 7 54 8 52 Group 3 10 3 19 7 41 8 38 10 25 10 57 columns 10 25 10 57 customizing 3 10 text format 9 14 inactive 3 10 Notes 3 9 3 13 7 47 8 45 text format 9 14 opening closing 3 12 order 9 13 Plate 3 9 3 15 printing order 9 12 rel

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