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MACSQuantify™ Software guide Basics of flow cytometric analysis

1. Import FCS file Export Sample Figure 7 55 Three files were selected for export 3 Configure the Export sample list box as follows Export sample list Options Region functions Feature functions Clipboard X File Location Public p Private Name srv2009 02 04_2062 052 ori xls V Conversions C Convert decimal point to comma C Transpose rows and columns C Reverse samples Cancel Options pd eset Check box to export data to the windows clipboard jal Fle Check box to export data to a Microsoft Excel file xls Location Files can be saved to a public or private location Name If File is checked enter the filename here taking care not to delete the Excel file extension xls Conversations the respective boxes to Convert comma to point in some languages numbers the decimal point is actually shown as a comma 153 Transpose rows and columns the columns and rows of the export table e g Excel sheet are inverted Reverse samples the order of the samples e g 1 2 3 etc are reversed Region functions Export sample list Options Region functions Feature functions Regions Functions f live af Name af srv2009 02 04_2062 054 pos gt lt of srv2009 02 04_2062 054 po T of srv2009 02 04_2062 054 po
2. 104 7 12 3 To perform backup to network drive 106 7 12 4 Configuring data backup settings administrators only 106 7 12 5 Configuring network settings administrators only 107 7 13 CONFIGURING THE DEFAULT USER INSTRUMENT AND SOFTWARE OPTIONS 107 7 13 1 Changing the default user options 107 7 13 2 Changing the default experiment options 110 7 13 3 Changing the default instrument options 111 7 13 4 Changing the default software options 113 7 14 DATA ANALYSIS IN CUSTOM MODE 3 373 373 3333355955 124 7 14 1 Creating a new analysis template or analysis window 124 7 14 2 Choosing a display format for plots histograms and statistics 126 7 14 3 Changing the properties of a plot histogram statistic or text table 127 7 15 WORKING WITH REGIONS OR GATES 135 Fit 3 1 DiAWING FEGIONS ASS 135 P1562 Gaung StralQies Sse seek eee eee eee eee ees 139 7 15 3 Changing the properties of regions 14 7 15 4 Post acquisition data analysis 143 Pid SD LIVE gal Es 148 fel NOSOP Gl E 149 7 16 GROUPING DATA POST ACQUISITION
3. 151 TANT EXPORT SAMPLES T e eee eee eee eee eee eee eee 152 8 Express mode 155 8 1 QUICK GUIDE TO THE EXPRESS MODE MAIN WORKSPACE 155 8 2 LOGIN TO EXPRESS MODE MMMMMMMMMMMMM 157 8 3 SWITCHING TO EXPRESS MODE FROM CUSTOM MODE 158 8 4 USING THE TOUCH SCREEN KEYBOARD ON THE MACSQUANT INSTRUMENT 158 8 5 DEFINING AN EXPERIMENT gt gt 35 9 159 Oi Jal RACK R eae ee ee See eee eee eee Se ee eee 159 8 5 2 Sample ID and Description 162 a o MOJE a aaa 162 8 6 WORKING WITH DATA FILES IN EXPRESS MODE 165 8 6 1 Introduction to file handling 165 8 6 2 Opening files 166 8 6 3 Saving files 167 8 7 DEFINING AN EXPERIMENT IN EXPRESS MODE A WORK THROUGH EXAMPLE 168 8 8 READING REAGENTS WITH THE CODE READER IN EXPRESS MODE 172 8 9 PRINTING IN EXPRESS MODE 3 37 3733 3959 rr 173 8 10 MACSQUANT INSTRUMENT DATA BACKUP IN EXPRESS MODE 174 8 11 EXITING FROM THE EXPRESS MODE 3 73 37 3755 95555 176 8 12 HOW TO CLOSE THE MACSQUANTIFY
4. Figure 7 20 Changing the default instrument annotations Here as an example on MACSQuant Instrument configuration 2 Enter desired alphanumeric text into the corresponding text box 3 Click Apply to implement changes Click OK close the window 112 7 13 4 Changing the default software options The following default software options can be modified keyboard timers acquisition settings export and backup settings and the default display settings for regions plots histograms and tables Each software option is briefly discussed below To assign default settings for the keyboard Note Feature only applicable to the MACSQuant Instrument This function is not available to MACSQuantify Software installations on personal computers B Options Figure 7 21 Changing the default keyboard settings 2 Use the drop down list to select a keyboard format The opacity can be modified as required 3 Click checkbox Show at start to automatically activate the touch screen keyboard at MACSQuant Instrument startup 4 Click Apply to implement changes Click OK close the window To assign default settings for timers Note Feature only applicable to the MACSQuant Instrument This function is not available to MACSQuantify Software installations on personal computers 113 1 Click Edit Options Software and Timers Files Tae Users Experiment Instrument Standby timer 120 min C Software Keyb
5. 1 2 So 1 amp 6 So o ow Figure 5 9 Chill 5 Rack was selected for multisample labeling 2 Left click once on rack coordinates Al and A2 oO 1 2 3 4 5 6 ey F EN ES k LA RAA B ES Tt ES g A N TA NZ NA A ISA N C i da a F A f Mest Mga Bek Sut Me Naot D rs ES Tt a aik N TA NS NLS NF O ONA NF Group Figure 5 10 Measure and select The settings for sample positions A1 and A2 may be modified e g a labeling strategy may be applied 3 Define the experiment settings for positions Al and A2 as shown in Figure 5 11 Samples Experiment Tools Channels Experiment Rack Chill 5 tube rack vi C File Trial 0 vja O Project MACS Control Cocktail CD14 F Sample ID Samples 1 amp 2 Description Human PBMC Flow rate Annotations Autolabel Settings Low Medium so High Pickup and measure CD45 VioBlue PE Cy Liquid sensor C Mix sample FITC CD15 4PC Mode Standard v CD14 PE APC Cy Uptake volume 50 ull cy PI Sample volume 200 pl _y i i Settings nats s Annotations Autolabel Settings Custom G Express F lt add Type Analysis vi E lt add gt Mode MC_CD1 4h O lt add gt C lt add gt C lt add E o i Reagent Time Titer rder IRT a MC CD14 Monocyte Cocktail human v 10 wia 1 10 via 5 vla i O 1 2 3 4 5 6 g Calibration SQuant Calibration Beads abg 5mL 30 viel i za 5 i Oo
6. E B Py cc BBs x ooo Heal a E O l S E e E l Samples Experiment Tools Channels Experiment BB Backup Ke Eg Rack Single tube rack Progress report File adm2011 07 21_22 INFO Initializing the backup system Project Sample ID Description Flow rate the Low wa Medium Pickup and measure Liquid s C Mix Mode Standa J Available drives Uptake vol eee Choose the destination for the files Sample volume gt Available drives so E Annotations dalla T i Available 3 GB Custom Exp C Instrument setting C Analysis template Gate CO Events y Data analysis mode Time ohare ne sta 17 34 ____oo100 _ ___ow_ _o e ay 10 Select all files or all data files when prompted 11 When back up is complete a dialog window appears 12 When the back up is performed again all previously backed up mqd files will be deleted if selected Choose delete cloned files All fcs files will need to be deleted manually through the copy function see below Transferring data using copy function 10 If using a USB thumb drive to transfer data insert into available USB port and wait a few seconds for the instrument to be recognized 11 Open the Copy function from the File menu 12 Choose the copy location from the available drop down menu USB thumb drive network location etc Copy 7 GB available lt MOSOFTSETUP E BB Public bac
7. 1 Insert the memory stick to the MACSQuant Instrument USB port or computer USB port Note If no external device is attached to the MACSQuant Instrument or personal computer the following error will be displayed External 2 Navigate to Edit gt Copy E g if you want to copy data files please highlight Data files on the left hand side Check the file you want to copy and select the destination You can copy all kind of templates data files log files and screenshots can be found under Other files for export Workspaces Instrument settings Experiments Reagents Analysis templates Data files and Other files e g screenshots or windows bitmap lt bmp gt files can be exported Bl Copy wie emt So am To x lt gt CY 261 MB available Workspaces Directory Name Size H C Gai Private gt Bechthold Original 004 fcs mad 2MB a7 7 ay Public Analysis templates X Gay 2011 07 15 Other files 1 files selected 2 MB Z m Log files 3 Click Copy 4 The file will be copied 7 12 Data backup and restore in Custom mode It is recommended that data is regularly backed up to an external location Data can backed up to a network drive USB memory stick or DVD Administrators can configure data backup settings Please contact your administrator for more information or refer to sections 3 and 7 6 of this software guide Note Before performing Backup ensure that the desired backup media is
8. 3 5 Getting started in the custom mode This section is intended to provide the user a quick overview of actions required fora quick start of the MACSQuant Instrument in the custom mode Some familiarity with the MACSQuantify Software is assumed 3 5 1 Turn on the instrument Switch on the MACSQuant Instrument by pressing the touch screen monitor while it is in standby mode The MACSQuant Instrument will automatically check and initialize the system following which the log in screen will be displayed 3 5 2 Login as administrator or custom user 1 Switch on the instrument 2 At startup a dialog box will appear prompting you to select your username and password The administrator admin account should be used for the first log in attempt MACSQuantify a on oS a U Serial No PC Version Registered for Jon Barbour Use E Password m Figure 3 6 Login for the first time as an administrator A dialog box prompts the user to confirm the password Note Custom users must only select their login name from the User drop down list No further action is required A password can optional be chosen 3 Select user account admin and enter a new password Select log in 4 After a successful log in the software will automatically prepare the instrument for analysis The status of this startup procedure is indicated by the 36 System setup dialog box After a few seconds the main screen will
9. 2 The MACSQuantify Software window will change to the Express mode Note If windows are active in the Custom mode e g analysis window the user will be prompted to confirm this action Click Yes to continue and No to cancel the switch Warning Note Any active work will NOT be transferred to the Express mode All data or settings must be saved before switching to Express mode 8 4 Using the touch screen keyboard on the MACSQuant Instrument The touch screen keyboard can be used to enter information into the Sample ID and Description fields Users may find it easier to use a conventional keyboard and mouse which may be connected to the back of the analyzer as described in the MACSQuant Instrument user manual To activate the touch screen keyboard perform the following 2 The keyboard popup window will appear 1 Click the Keyboard icon 3 Click the Keyboard icon once again to close touch screen keyboard 158 8 5 Defining an experiment Note All experiment and analysis templates are defined by the Administrator or a Custom user The Express user may apply these settings to newly acquired data but can not create analysis templates In order to perform an experiment the following criteria must be defined 8 5 1 Rack Five different kinds of sample tube racks are available see Table 8 2 for details The Chill Racks Chill 5 Chill 15 Chill 50 and Chill 96 must be used with the MACS MiniSampler T
10. 7 6 Data backup and file transfer There are two ways to transfer data from the MACSQuant Instrument to a remote storage location network folder USB external hard drive DVD etc O 1 Back up using the icon for backing up data to a network location external hard drive USB drive or DVD 2 Copy from file menu for transferring data to a network location external hard drive or USB drive Backing up data from the MACSQuant Instrument 1 Provide the full network path for data back up Edit lt Options lt Files Asa custom user a private back up location can be defined 2 When ready to back up your data files click on the Q icon 3 If a network folder is the back up destination enter password when prompted File Edit View Mode Analysis Window Help ooo p admin on MQ2209 SS ESSE rr SSO eee e e a a po EM fe tl a ERRA Samples Experiment Tools Channels Experiment BB backup Eg Rack Single tube rack Progress report File adm2011 07 21_22 INFO Initializing the backup system Project Sample ID Description 7 Flow rate Low a Medium Pickup and measure Liquid sensor C Mix Mode Standa i Available drives Jeg Uptake volume EO 7 Choose the destination for the files Sample volume ee x Annotations Autolabel f Custom O Ew vailable ook ancel C Instrument setting Available drives se F C
11. Access Instrument Using the drop down list set the user access settings for each of the following criteria Experiments None User access is unavailable Reagents Read User access is restricted to read files only Analysis l Read amp Write Full user access is available i e Data files read and write data to this folder These options are available for Public and Private accounts Note the data files can only be stored in one location either Private or Public Password Required Activate checkbox to ensure password restricted access to the account Activate checkbox to reset an associated password The user will be prompted to enter a new password at the next log in attempt Table 3 2 Creating a new user setting user properties 4 Click OK to save the new user settings PR List of Users User i Initials Group Rights Directory Public Private Fassword F adm admin AXE C cap useradmin IREAD IRE d peg ith empress E Ccap uzerjohn smith ierad IERAD peg Figure 3 5 An Express password protected user account was created for John Smith JS 5 To modify or delete the user account click Properties or Remove respectively Note If the user is set as an Express mode user the user will be automatically logged into the Express mode If the user is set as a Custom mode user the user will be 35 automatically logged into the Custom mode window This same applies to administrators
12. A final population of enriched activated IL 17 secreting CD4 T cells are shown MiN2009 02 27_2053 021 P1 P2 P3 CD154 APC A 1e2 1e3 1 1e0 lel Anti IL 17 PE A Figure 7 44 The region P1 P2 P3 was displayed using the axis Anti IL 17 PE versus CD154 APC 10 Click to expand the gating strategy in the Samples menu i e MIN 2009 02 27 2053 005 100 0 eeds4 138 IL 17 Analysis_complete mq admin File Edit View Mode Analysis Window Help aaa dododd daela Ouig lela aio Samples Experiment Tools Channels Sample DEK Eg MiN 2009 02 27_2053 021 PIPAP3 1 81 T Figure 7 45 An overview of the entire gating strategy Note The regions in this gating strategy do not have the same hierarchy Regions are created within regions leading to subdivisions of gates regions Samples Experiment Channels 0 WiN 2003 02 27 2053 021 i P3463 17691 1328 1328 11 To save the gating strategy as an analysis template e Ensure that the MACSQuantify Software is in Analysis mode Al e Click and e Choose the file location Public Private or External e Name the file Click Save 7 15 2 Gating stratgies Note For background information about drawing regions gating with MACSQuantify Software refer to section 7 15 1 139 Classical hierarchical gating In section 7 15 1 an example of a hierarchical gating strategy is given In this classical strategy gates are
13. Analysis template Cell Cycle odora cells w Gate ve v Events 10000 a 5 Ensure that the correct instrument settings are loaded and that compensation is correctly performed Note See section 4 1 for information about performing instrument calibration refer to section 4 2 for information about performing compensation 150 6 Ensure that enough sample reagents and buffers are provided 7 Click on the Start Measurement button gt The MACSQuant Instrument will commence sample uptake and measurement 8 Draw regions on the plots as described in section 7 15 1 Note Click to delete all events displayed on a dot plot i e to refresh the plot 9 Save the Analysis template for future use 7 16 Grouping data post acquisition What is the benefit of sample grouping The maximum sample volume that can be acquired in a single step by the MACSQuant Instrument is 450 uL There are occasions when the sample size is of course greater aliquots of the sample must therefore be spanned over two or more tubes By grouping these samples the acquired data will be consolidated into a single file on the hard drive which can also be analyzed in a single data file or analysis plot This can be easily accomplished by grouping sample prior to data acquisition Refer to section 5 5 3 for more details If grouping was not performed prior to data acquisition it is still possible to group samples post acquisition A D a
14. Calibration Chrl alt c Command Undo Redo Copy page Copy plot Delete region Ellipse Rectangle Polygon Quadrant Interval User settings Instrument settings Rack Reagents Options Configuration Calibration Description Undo or redo the most recently performed action e g undo create region Copy the entire analysis window to the clipboard All dot plots and tables are copied Copy a single selected plot histogram highlighted in green Delete a region that was created in a dot plot Create the mentioned geometric shape in a plot Create and or modify user account settings Administrator only Modify the instrument settings comprising Channel Compensation and Custom To edit the sample rack settings To open the Reagents dialog box and modify reagent settings To modify User Experiment Instrument and Software options Only available as administrator When using MACSQuantify on the PC use this option to designate the optical configuration that data files were collected with To view and or modify instrument calibration settings 30 View M Mode Analysis Window Command Description Hardware Hardware To view the hardware settings comprising Fluidics Sample Experiment table uptake unit Lasers and detectors Camera and System settings Experiment table Provides a tabulated overview of experiment details Acquisition
15. Figure 5 4 The MACSQuant Instrument reagent management window e Pos Use this checkbox to assign reagents to rack positions R1 R2 R3 R4 S1 or S2 o RI R4 positions are located on the MACS Reagent Rack 4 S1 and S2 positions are denote as Special positions where the Running Buffer is taken directly from the buffer bottle e Category Reagents are categorized according to species conjugated fluorochrome and purpose The current categories follow o Calibration MACSQuant Calibration beads for calibration of the instrument settings o Species and conjugated fluorochrome e g Human APC Mouse PE o Isotype control isotype control antibodies are raised against non mammalian epitopes and can be therefore used as a negative control for non specific binding 67 o MACS Comp Reag MACS Compensation Reagents These reagents are used to correct the inherent spectral overlap between excitation and emission wavelengths of fluorochromes o Universal for generic labeling strategies using Tags such as biotin His or propidium iodid e Reagents A drop down list of available reagents is displayed in accordance with the selected category for example the following reagents are available for the category Human Cocktails MC CD14 Monocyte Cocktail human MC CD19 B Cell Cocktail humar MC CD3 Pan T Cell Cocktail human MC CO34 0017 33 Cocktail human MC CO34 001733 Control Cocktail humar MC COS T Cell Coc
16. PE Medianneg PE PE Medianneg gt PE Medianpos PE Medianneg lt PE Medianpos Medianpos Figure 4 5 FITC single stained cells 46 4 3 General guidelines for proper compensation For proper compensation the use of single stained controls is absolutely necessary These single stains can be cells stained compensation beads or antibody capture compensation beads but there are several points to take into consideration when setting up and choosing single stained controls for compensation 1 2 3 4 5 6 7 Compensation should be performed using the same fluorochrome that will be detected in the experimental panel This means that if FITC is used in the experimental staining panel FITC must be used to set the compensation Do not substitute with another fluorochrome that is detected in the same channel such as GFP or Alex fluor488 The positive and negative populations within the sample must have the same autofluorescence or level of background fluorescence If utilizing a fluorochrome conjugated to a different antibody for setting the compensation adjustments the fluorescent intensity level of this staining should be as bright as or brighter than the signal from the experimental panel All changes to voltages of fluorescence PMT detectors must be made prior to adding compensation adjustments Changing PMT voltages after compensation will change the spill over detected and will require
17. The settings for regions and functions are identical to those for dot plots Several regions gated cell populations can be displayed ona single histogram In the following example four regions P1 P2 P3 and P4 were color coded and displayed Overlay _2063 015 pos P4 300 v Population 1 2008 1 2 10_2063 015 pos P1 opulation stv g pos m 250 Population 2 stv2008 12 10_2063 015 pos P1 P2 m 200 Population 3 stv2008 12 10_2063 015 pos P1 P2 P3 150 Population 4 srv2008 1 2 10_2063 015 pos P3 P4 m si 1 0e 21 0e 11 0e01 0e11 0e21 0e3 VioBlue A Table 7 2 An overview of the Properties settings for histograms 132 Overview of the Properties settings for the statistic option A Statistic text box is composed of a Header and a Table s1 2008 12 10_2063 015 pos P1 P2 P34 P4 2008 12 10 16 42 Descr File am 2008 1 2 10_2063 015 pos mgd Sid pog 16 I YioBlue Mean r2 bo ra 16 53 96 g2 33 YioBlue StdDey 20 97 au r0 PE A Mean PE A StdDev 21 36 20 83 af 37 96 Table Figure 7 39 A header and a table comprise a statistic text box A variety of display settings for the Statistic option can be modified using the tb i 7 Properties option from the drop down menu 1 Click the icon located adjacent to the Statistic text box 2 Click Properties Properties The properties window will appear To change the contents of the statis
18. Figure 5 7 Configuring a sample rack using the MACSQuant Instrument touch screen e When arack position is selected for the first time by a single mouse click the following status is reported 72 e By re selecting the same rack position the status changes from EVA J An explanation of the various rack configurations for single sample positions is given by section 5 4 User action with left mouse Effect DACIE button or fingertip action on touch screen Default open circle indicates no operation Clear Single click on circle single Closed green circle with orange rim Sample selected finger touch for measurement Orange circle indicates that the sample is selected and any alterations made to the measurement strategy e g labeling will only apply to sample positions with this designation Double click on Closed green circle Sample selected for measurement circle double finger touch Closed blue circle Measurement in progress Closed gray circle Measurement finished Closed yellow circle Processing of sample has commenced e g sample has been labeled and incubation is underway Table 5 3 Summary of rack configurations More than one rack position can be selected at once An entire rack row or column can be selected or deselected Furthermore samples can be grouped together using the Group function After sample acquisition grouped samples can be analyzed together in a single dataset analysis window 73 Use
19. Instrument settings are compensation and calibration parameters for the MACSQuant Instrument These parameters are important for data analysis and are vital to maintain standardized results over time and from instrument to instrument The MACSQuantify Software can open and save instrument settings These settings can be applied to acquired data and thus this useful feature allows users to perform recompensation after data acquisition The Instrument settings may be saved but not opened in Express mode Experiment definitions can be saved for future use Reagent type and corresponding Reagent Rack 4 positions sample rack type and corresponding Chill Rack sample positions the analysis mode and sample processing definitions e g labeling strategy comprise experiment definitions Analysis templates are predefined analysis layouts for data acquired by the MACSQuant Instrument The templates are creating by defining a gating strategy with associated plots histograms tables and statistics Administrators and Custom users can customize and save templates for reuse Express users cannot create or modify Analysis templates Data files can be saved to a Public Private or External file location by all users MACSQuant Data Data MQD is the standard file handling format however the MACSQuantify Software can also import Flow Cytometry Standard FCS file types 7 2 File structure Defined files are saved with the cap folder on your harddri
20. Mode Note Contact your MACSQuant Instrument administrator if the desired template is not available Templates can only be created and managed by administrators or Custom users Selecting Express mode programs 1 Select Analysis from the Mode drop down menu 163 2 From the drop down box select the desired Analysis criterion Find an example list of options in Table 8 5 Corresponding graphic Option Description Count To perform absolute cell counting MC_CD14_h Evaluation of MACS Cell Separations using CD14 MicroBeads human or the Monocyte Isolation Kit Il human 130 092 859 MC_CD_19_h Evaluation of MACS Cell Separations using the B Cell Isolation Kit human 130 092 860 MC_CD_34_h Evaluation of MACS Control MC CD34 Stem Cell Cocktail human 130 093 427 MC_CD3_h Evaluation of MACS Cell Separations using the Pan T Cell Isolation Kit Il human or CD3 MicroBeads human 130 092 881 MC_CD4_h Evaluation of MACS Cell Separations using the CD4 T Cell Isolation Kit I human or CD4 MicroBeads human 130 092 914 MC_CD8_h Evaluation of MACS Cell Separations using the CD8 T Cell Isolation Kit human or CD8 MicroBeads human 130 092 912 Table 8 5 Examples of options available for performing cell analysis in Express mode Note Updates to the analysis mode menu are performed regularly additional options may be available Details on Express mode programs can be
21. P2055d P3005n CP1515n PC2025n Hp Officejet Pro 8000 Note For a complete list of printers compatible with the HP Universal Print driver please visit www hp com go upd Please note the only the above mentioned printers have been tested with the MACSQuant Instrument Note It is also possible to print to a network printer Please contact your MACSQuant Instrument administrator or Miltenyi Biotec Technical Support for more information To print active workspaces 1 Open the desired workspace or analysis window 65 2 Click 3 Select the desired printer Click Print The printer can be networked to or directly connected to the MACSQuant Instrument or to the PC running the MACSQuantify Software The active workspace is printed as shown below Date 2008 16 23 Operator admin Version 1 2 0902 R arie rigig a 6 pal AFE EA TO 17 DT Da Te et et Sees es ee Joie E PES me S110 E16 pee Legend Pagan Prpdsten Geurl m Leo ache Tat g Pies Yebe aubrey ies 117176 PTEI Dreikas ce ray ot ata er rra Results pos 16 lag Freq N Conc Umi ee U Viable leukocytes warn A S 22464005 i Viable CD14 monocytes wenn 9 7 1 56E 005 pete ee 1T 1 T s me Figure 5 2 Half size example print out of data analyzed by the MACSQuantify Software in Custom mode 5 3 Reagent management MACS Reagent Rack 4 in combination with the MACS MiniSampler is used for autolabeling of cells
22. accessible to the MACSQuantify Software Backup media The backup procedure OQ searches for backup media in the following order 1 A designated folder located on a local area network this must be setup by an administrator with assistance from Miltenyi Biotec Technical Support 2 Amemory stick attached to the USB port on the MACSQuant Instrument 3 Arewritable DVD 4 If none of the above are found the MACSQuantify Software reports an error No valid backup devices found Progress report INFO Initializing the backup system INFO Checking files Cannot initialize the disc burner If there are errors reported resolve them and try it again 102 7 12 1 To perform a backup to a rewritable DVD 1 2 3 4 5 6 7 8 Ensure no USB stick is installed and that no network drive has been defined as the default location for backup files Please contact your administrator for further advice Insert a rewritable DVD into the MACSQuant Instrument DVD drive Only DVD R or DVD RW media may be used DVD RW and CD media types are not currently supported Wait for 10 20 seconds after inserting the DVD into the drive Click the backup icon located on the top menu bar The files will be written to DVD Note Depending on the amount of data the backup procedure may take several minutes When the progress bar displays 100 the MACSQuantify Software will verify the data once again this may take
23. found at www macsquant com 3 Click Start Measurement gt 164 8 6 Working with data files in Express mode Refer to the sections Opening files and Saving files for immediate instructions or handling these file types If you are unfamiliar with the user interface or options associated with handling files read the following information at 8 6 1 Introduction to file handling 8 6 1 Introduction to file handling This section describes how data files can be opened saved and backed up in Express mode Data files may be stored to and therefore opened from a Public Private or External file location Public L Private External e Public files are located on the local hard drive of the MACSQuant Instrument or personal computer and are accessible by all users e Private files are located on the local hard drive of the MACSQuant Instrument or personal computer and are only accessible by the logged in user account e External files are located on an independent file storage device which is connected to the MACSQuant Instrument or personal computer via the USB port i e a memory stick The default window for saving and opening data files is composed of the following tabs Note The availability of these tab options is dependant on the user profile Custom user Express user or Administrator and whether data settings are being saved or opened Tab option Description Instrument settings
24. srv2009 02 04_2062 052 ori E MC_CD4_T_Cell_Cocktail_h stv2009 02 04_2062 053 neg E MC_CD3_Pan_T_Cell_Cocktail_h stv2009 02 04_2062 054 pos fm MC_CD19_B Cell Cocktail_h E MC_CD14_Monocyte_Cocktail_h GaLz E CD34cocktail hlog E CD34_CD133 cocktail log5 E CD34_CD133 cocktailhlog E CD34 Stemcell log5 E 2009 09 01_Calibration C 2009 04 28 Figure 7 7 Highlight the desired tab to view available files The Experiment left or Data files right tabs were selected in this example Note Multiple data file types can be selected and opened at once right To open workspaces 1 Highlight the Workspace tab on the Open window 95 2 Highlight the file location External to open files from external media e g a USB memory stick Files are opened from the default Private location 3 Select the file type and click Open L To open instrument settings 1 Highlight the Instrument tab on the Open window 2 Highlight the file location Private Public or External 3 Select the file type and click Open L Instrument settings Calibration for cell cycle CD14 Experiment Setting CD14 Exptl To open experiment definitions 1 Highlight the Experiment tab on the Open window 2 Highlight the file location Private Public or External 96 3 Select the file type and click Open L Public Private wal External Workspaces Directory Experiments experiments
25. 1 2 10_2063 013 ori P 2008 1 2 10_2063 013 0ri P noo Canoe TE Description All events are displayed on a dot plot or density plot 50 25 5 2 or 1 percentile values of the total events can be displayed on a dot plot or density plot This is useful when too many events have been acquired and the displayed plot histogram is saturated A fixed number of events can be displayed on a dot plot or density plot Numbers can be entered directly into the field or by using the arrows The y and x axes scales of a dot plot and density plot or the x axis scale of a histogram can be configured as follows As required automatically configured lin linear scale log2 5 logarithmic scales hlog hyperlog scaling All regions gates are shown on the chart Only a select region gate is shown on the chart No regions gates are shown on the chart Regions gates can be displayed vw or hidden A by selecting or deselecting a region using the left mouse button or by touching the display with your fingertip Functions for a region can be displayed w or hidden K by selecting or deselecting the function using the left mouse button or by touching the display with your fingertip The following functions can be changed for plots and histograms Name Region name e g P1 130 Events in selected region are displayed as a percentage of the total
26. 4 130 094 542 was used for the analysis of viable human IL 17 secreting leukocytes from PBMCs Enriched human IL 17 secreting cells were labeled as follows 135 IL 1 7 Detection Antibody PE conjugated fluorochrome CD4 marker FITC conjugated fluorochrome CD154 marker APC conjugated fluorochrome Note For background information about drawing regions gating with MACSQuantify Software refer to section 1 2 8 To create draw a region 1 Open a data file or simply draw a region around events that are being acquired O 2 3 4 by the MACSQuant Instrument in real time Click File Open and highlight the data file s to be analyzed Click Open Alternatively Select the Sample tab and right click Open in the sample window Samples Experiment Tools Channels Statistic Cels Sample live Add Import FCS file Export Sample Resample Export sample list Highlight the data file s to be analyzed Click Open Click to open an analysis window Select the desired plot layout In this case a Plot4 layout was chosen Select the plot of interest A green border indicates the chosen plot Double click on the opened data file this is not necessary when acquiring data in real time Samples Expenment Tools Channels Sample Statistic Cels ie g i 73463 Figure 7 40 Left The data file Wikies was opened in
27. 5 Click Apply and OK 7 15 3 Changing the properties of regions 1 Click on the region of interest Note To select a region click on the region name using the Sample menu EZE or directly click on the region geometric region displayed on the plot File Edit View Mode Analysis Window Help admin Ce ENE doada Haaa EY EE O VS cE live amp stv2008 12 10_2063 015 pos 2 OPI BO P2 a OP3 P4 cell cycle 001 2 To change color region name and or define the region as NOT e Right click on the region name displayed in the Sample menu or right click on the activated gate in the dot plot 141 e Select Region properties File Edit View Mode Analysis Window Help gla 90943 sao a Guisisld iziao Samples Experiment Tools Sample So Cells amp stv2008 12 10_2063 015 pos ua 14887 OPI 91 0 13550 amp OQ P2 75 1 11176 2 OP3 53 6 7975 So A A Import FCS file Fp ese Remove cell cycle 001 _ e Select the required option s Click Apply and OK Region properties 3 To change region functions e Click the icon which is adjacent to the plot of interest e Select the required options Click Apply and OK v os Beaune ht LA ee er r ee Pe 2 eed Qt ee ee Me ed 4 Inthe example below region P3 was displayed in blue and set as a NOT region Region properties 142 7 15 4 Post acquisition data
28. 5 DEFINING AN EXPERIMENT WITH MULTISAMPLE PROCESSING A WORK THROUGH EXAMPLE 76 5 5 1 Background 76 5 5 2 Rack configuration and sample definition 76 5 5 3 Rack configuration and sample grouping 79 6 MACS Pre enrichment with MACSQuant Instrument 82 6 1 UTILIZING THE MACSQUANT COLUMN FOR PRE ENRICHMENT A WALK THROUGH EXAMPLE 84 7 Working with data files in Custom mode 88 7 1 INTRODUCTION TO FILE HANDLING 73 99 9 88 Fal FILE STRUCTURE 2325230 6 ee ee ee ee eS ee eee ees 89 7 3 OPENING DA TA FILES Se ee ee ee ee ee Se ee ees 90 7 4 ADDING DATA FILES 2 22336 Soe ee ee eee See ee eee eee Seca eeeS 9 7 3 IMPORTING FCS GILES 32a ee ee eee ee ee Se ee ee ee ees 9 7 6 DATA BACKUP AND FILE TRANSFER gt gt 7 9 9 9 92 7 7 VIEWING ANALYSIS TEMPLATE USED DURING DATA ACQUISITION 94 7 0 OPENING FILES 2 26S ie ei ee eee ie ee eee eee se eees 95 7 9 SAVING FILES 22S Se ee Se ee eee ee eee 98 71 0 IMPORTING FILES Sa eee ee ee ee ee eee 99 TU EXPORTING FILES eee ee ee ee eel 101 7 12 DATA BACKUP AND RESTORE IN CUSTOM MODE 102 7 12 1 To perform a backup to a rewritable DVD 103 7 12 2 To perform backup to a USB memory stick
29. Annotations Autolabel and Settings Mode Analysis Window H Command Description les Dot plot Dot plot Density plot Click icon to change the Histogram Statistic presented data format into le r E uc Text another format e g from a dot plot into a histogram Mh Histogram Multilayer mode View data in a multilayer Statistic format Text Im Multilayer mode Analysis Window Help Command Description Analysis mode i Previous sample Analysis mode Activate Analysis template tool Previous sample Next Scroll through analysis Next sample sample windows in a reverse and forward direction 31 Window Re New analysis window E T Clone window Command Description New analysis window Open a new analysis window using predefined templates E Close Clone window An exact clone copy of the analysis window is made This X close all includes gating strategies and open data files Previous window Close Close all Close a selected analysis deena window close all analysis EXE WIATA gt windows Previous window Next Sequentially scroll through window analysis windows i e previous and next analysis window Help Command Description Help Open help Help Open the preinstalled help file Info Info Information about current software version 3 2 Options When using MACSQuantify Software users can set up or change default settings of cert
30. CD34 Stem Cell Analysis Instrument settings i x age Analysis templates Data files Experiment PBMC Analysis B To open analysis templates 1 Highlight the Data files tab on the Open window 2 Highlight the file location Private Public or External 3 Select the file type and click Open L Public Private External Delete Workspaces Directory Analysis templates E analyses BRE Instrument settings Data files Template CD14 To open data files 1 Highlight the Data files tab on the Open window 2 Highlight the file location Private or Public 97 3 Select the file type and click Open L m Public sa Private Delete Workspaces Directory Data files 9 data im adm2009 09 01_2022 001 E PBMC jadm2009 09 01_2022 002 E MC_CD4_T_Cell_Cocktail_h jadm2009 09 01_2022 003 3g MC_CD3_Pan_T_Cell_Cocktail_h E MC_CD19_B_Cell_Cocktail_h I MC_CD14_Monocyte_Cocktail_h Experiments 0 IL 17 E CD34cocktail hlog 59 CD34_CD133 cocktail log5 E CD34_CD133 cocktail hlog E CD34 Stemcell log5 E 2009 09 01_ Calibration E 2009 04 28 Instrument settings Reagents Analysis templates Files adm2009 09 01_2022 001 External Directory Workspaces E ee Expt Instrument settings Experiments i Reagents KT Analysis templates Workspace Figure 7 8 The Save window Custom users and administrators are able to sa
31. Cable connecting the fluid sensor to the MACSQuant Instrument The sensor cables are color coded blue for Running Buffer green for Washing Solution black for Storage Solution and red for waste 186 Front panel MACS Cell Enrichment Unit MACS MicroBeads MACS Technology Negative fraction PE Positive fraction Cell enrichment Syringe pumps Touchscreen Tubing connector Tubing system Chill Racks Uptake needle Waste container The front panel opens sideways giving access to the MACSQuant Instrument Column pumps valves washing station and tubings Black cover surrounds the area enclosing the magnet The magnet cover is located in the center of the fluidics system and has a slot for the insertion of the MACSQuant Column Superparamagnetic particles conjugated to antibodies for magnetic labeling of cells or biomolecules Technology for immunomagnetic labeling and subsequent separation of cells or biomolecules in a high gradient magnetic field Sample fraction containing the cells not magnetically labeled During MACS Separation these cells are not retained on the column and pass directly through the column while the column is in the presence of the MACS Magnet R Phycoerythrin Sample fraction containing the cells labeled with MACS MicroBeads after MACS Separation These cells are retained on the column while the column is placed in the magnetic field The cells are eluted from the column a
32. Cocktail human 130 093 427 1 3 MACS Cell Separation MACS Cell Separation i e the magnetic separation of defined cell populations using MACS Technology is widely regarded as the gold standard in cell separation MACS Technology is based on the use of MACS MicroBeads MACS Columns and MACS Separators strong permanent magnets MACS Technology can be used for the targeted cell enrichment or depletion of cell types or populations through their expression of particular surface antigens MACS Technology provides the means for the pre enrichment of rare cells for subsequent flow cytometry analysis In a first step surface antigens are magnetically labeled in a highly specific manner with monoclonal antibodies coupled to MACS MicroBeads MACS MicroBeads are superparamagnetic particles of approximately 50 nanometers in diameter comparable to the size of a virus MACS MicroBeads are not known to alter the scatter properties of cells in the flow cytometer or influence the light microscopic appearance of the cell They form a stable colloidal suspension and do not precipitate or aggregate in magnetic fields MACS MicroBeads are composed of a biodegradable matrix made of iron oxide and polysaccharide hence it is not necessary to remove them after the separation process saving hands on time MACS MicroBeads do not alter the structure function or activity status of labeled cells and they are not known to interfere with subsequent experiments F
33. Experiment Public Private Read amp Write d Read amp Write x Read amp Write z z Rosie Reagents Read x Read amp Wrte x T 5 x Read amp Write x Figure 7 17 Changing default file paths for user groups 2 Use the drop down list for each access category to modify the read and write privileges as required 3 Click Apply to implement changes Click OK close the window The defaults for the user setting dialog will be set instrument settings Read Read amp Write x Experiments Read Read amp Write w Analysis Read Read amp Write xw Data files Read Read amp Write x Password 7 Required C Reset kE 7 13 2 Changing the default experiment options The default Experiment settings can be modified using the Experiment options menu 110 To assign default experiment settings 1 Click Edit Options and Experiment I Options ec mS Files o Ta l Users Experiment Access Flow rate Low Instrument Annotations SER Software Liquid sensor Mix sample Keyboard z ioe Mode Standard Acquire ak 10041 Export Uptake dj Statistic m a Volume 200 u GQ FCS Backup Regions Event limit 10 000 G2 Windows OK Cancel Figure 7 18 Changing default experiment settings 2 Modify the default values for Flow rate Mode Uptake Volume and Event limit as required Note It is n
34. First name Joe Last name Bloggs Reauired Country City USA Email address fjoe bloggs laboratory com Reauired Phone number Country region Number with area city code 1 99999999999 Message Question I would like assistence with 3 Click Submit 4 Live support will commence 180 11 Troubleshooting The instrument automatically analyzes the functionality of several hardware components during the instrument initialization procedure An error message will appear if action is required by the user Please refer to the MACSQuant Instrument user manual for help with dealing with certain error messages otherwise contact Miltenyi Biotec Technical Support If problems appear which are not indicated by a warning or error message that might occur during measurement cell separation or rinsing programs contact Miltenyi Biotec Technical Support 181 12 Hardware monitor Using the hardware monitor the current hardware status of the MACSQuant Instrument can be assessed at any time The hardware pages can provide additional information on the status of the instrument in the case of error messages appearing on the screen Never run the instrument if any errors are indicated within the hardware monitor 12 1 Hardware monitor window The Hardware Monitor window can be accessed under the View menu The window contains five tabs each displaying the hardware components involved in the respective processes Pleas
35. High Flow rate C S00 s gal o 2000 5 Pickup and measure 1 Optional Click the Mix sample checkbox Ll Mis sample to premix the sample before sample pickup data acquisition and analysis 61 2 Select an analysis mode from the Mode drop down list Standard See Table 1 1 for details about each option Uptake volume 3 Enter the Uptake volume and Sample volume 72 volume A maximum sample volume of 5 mL and a maximum uptake volume of 450 uL can be entered Annotations tab 1 Optional If required modify the annotations for the fluorescence channels The default settings are shown below Annotations Autolabel Settings WT CD 45 VioBlue B4 PE Cy B1 FITC Al CO15 APC B2 CD14 PE R2 APC Cy Autolabel tab 1 Optional If autolabeling is required add the relevant reagents by clicking on an lt add gt checkbox Annotations Autolabel Settings E lt add gt lt add gt lt add gt lt add gt lt add gt lt add gt lt add gt lt add gt lt add gt lt add gt 2 Click an available checkbox 3 Select the reagent reagent rack position incubation time titer and order from the drop down list 62 Pos Category Reagent Time Titer Order RI Human PE CD45 PE human wy 10 va ttt val 5 val R2 c MACSQuant C Beads a J vla F100 vf CS vf R3 3 g L 0 vja 1 100 vja 5 v
36. Print The printer can be networked to or directly connected to the MACSQuant Instrument or to the PC running the MACSQuantify Software 4 The current analysis page is printed as shown below 173 Date 2003 10 16 paar admin vasim 2 0 06G0 4 ak it ee a ra l A HPE GSA S 20 Si T note iis iis i 1i Teed Pac nt L 77 PG A Wiha SE oe erie its eee bea Oa ra GOS AA or FT G A 1 oe D1 1 Sa 1 eT 1 tee 1a Lied 1 LT 1 Sal 12s 1 tet 1 fe Sni TAA A081 L 17 PE Figure 8 19 Half size example print out of data analyzed by the MACSQuantify Software in Express mode 8 10 MACSQuant Instrument data backup in Express mode There are two ways to transfer data from the MACSQuant Instrument to a remote storage location network folder USB external hard drive DVD etc 3 Back up using the icon for backing up data to a network location external hard drive USB drive or DVD 4 Copy from file menu for transferring data to a network location external hard drive or USB drive Backing up data from the MACSQuant Instrument 7 Provide the full network path for data back up Edit lt Options lt Files Asa custom user a private back up location can be defined 8 When ready to back up your data files click on the icon 174 9 If a network folder is the back up destination enter password when prompted File Edit View Mode Window Help admin on MQ2209
37. SOFTWARE 177 9 Shutdown of the MACSQuant Instrument 178 9 1 MANUAL SHUTDOWN 3 3 999 9 rrr rrr 178 9 2 AUTOMATED SHUTDOWN 33 399 rrr rrr 178 10 MACSQuant Live support 180 11 Troubleshooting 181 12 Hardware monitor 182 12 1 HARDWARE MONITOR WINDOW 33 7 3333399999 rrr 182 13 14 15 Technical Support Limited warranty Glossary Introduction 1 1 Purpose The MACSQuant Instruments are benchtop flow cytometers that have been specifically designed for the rapid simple and automated fluorescence analysis of single cell suspensions The MACSQuant Instrument also facilitates the absolute quantitation of cell populations and has a processing rate of up to 10 000 events per second The instrument was designed for use with MACS Cell Analysis and MACS Separation Reagents in research applications though common fluorochrome conjugated antibodies and fluorescent reagents from other suppliers can also be used The relatively small footprint of the MACSQuant Instrument 60x35x40 cm in comparison to other commercially available flow cytometers makes the instrument
38. a few minutes to complete Note At this stage data will NOT be deleted from the MACSQuant Instrument the data is only copied to DVD Insert the backup DVD into the destination DVD drive of an independent personal computer on which the MACSQuantify Software is preinstalled This computer can be used for data analysis Start and login to MACSQuantify Software on the personal computer Click restore ee Note MACSQuant Instrument data will be copied to the local drive of the personal computer After a successful data transfer the copied data will be marked as successfully copied on the DVD Note When performing a future data backup on the MACSQuant Instrument ensure that this backup DVD is used 103 Performing subsequent MACSQuant Instrument backup 1 Insert the designated MACSQuant Instrument backup DVD 2 Click backup Note The MACSQuantify Software will identify data marked on the DVD as successfully transferred to another computer This corresponding data will be deleted off the MACSQuant Instrument hard drive and DVD before continuing with the backup procedure o 3 After backup is finished remove the DVD from the drive 4 Transfer the data to an independent personal computer as described above H i Data written to DVD aa _ but NOT deleted a l from the lt MACSQuant Click k N Analyzer i Backup Insert unused icon Fa Backup is performed DVD R
39. and P3 regions under the region functions Select to display the median of all the spill over fluorescence channels e g For FITC spill over display V1 V2 B2 B3 B4 R1 and R2 although not all require compensation 16 Resume measurement by clicking on the pause button 17 Enter values in the box within the compensation matrix by using the associated slider For example compensating FITC signal from the PE detection channel the fourth box down within the third column should be adjusted 18 Values should continue to be adjusted until the Median fluorescence values for the positive and negative populations are equal for the spill over channel 19 Adjust values for other spill over channels as necessary 20 Once compensation is adjusted for this fluorochrome repeat for all additional fluorochromes 4 5 2 Automated compensation using the Express protocol CompensationMultiColors Materials required 1 Single stained controls representing all fluorochromes to be used in the experimental staining panel Ensure that there is a comparable positive and negative population for setting compensation 2 12x75 mm round bottom tubes or 96 well plate 3 MACS MiniSampler 4 Chill 5 or Chill 96 Racks Set up and perform automated compensation process 1 Load in the instrument setting for compensation 2 Select the Chill5 rack from the Experiment tab 3 In the large Chill 5 window choose the appropriate number of positions to mat
40. appear in Data analysis mode 3 5 3 Activate the touch screen keyboard on the MACSQuant Instrument The MACSQuant Instrument is equipped with a keyboard as well as with a touch screen keyboard To activate the touch screen keyboard perform the following 1 Click the Keyboard icon 2 The Keyboard popup window will appear 3 Click the Keyboard icon once again to close touch screen keyboard 3 5 4 Check the fluid levels Fluid levels of the buffers and solutions need to be checked before each application Check that sufficient Running Buffer and Washing Solution are present in the containers for the measurement minimum 150 mL of each Replace any of the solutions whose levels are low Also check that the waste container is empty Please note the bottles are only illuminated after the system has been placed into acquisition mode If any buffers or solutions need to be changed 1 Press the main instrument control icon to set the instrument into Acquisition mode This will initiate the priming of all of the fluidics turn on the illumination LEDs for the four buffer bottle and turn on the lasers Each time you select the main instrument control a dialog box will appear providing two of three 37 options Analysis mode Acquisition mode or Instrument off File Edit View Mode Analysis Window Help Ere Nl SEE ci Quigjelel Experiment mim File adm20
41. are compensation and calibration parameters for the MACSQuant Instrument These parameters are important for data analysis and are vital to maintain standardized results over time and from instrument to instrument The MACSQuantify Software can open and save instrument settings These settings can be applied to acquired data and thus this useful feature allows users to perform recompensation after data acquisition The Instrument settings may be saved but not opened in Express mode Experiment definitions can be saved for future use Reagent type and corresponding Reagent Rack 4 positions sample rack type and corresponding Chill Rack sample positions the analysis mode and sample processing definitions e g labeling strategy comprise experiment definitions Analysis templates are predefined analysis layouts for data acquired by the MACSQuant Instrument The templates are created by defining a gating strategy with associated plots histograms tables and statistics Administrators and Custom users can customize and save templates for reuse Express users cannot create or modify Analysis templates Data files can be saved to a Public Private or External file location by all users MACSQuant Data MQD is the standard file handling format however the MACSQuantify Software can also import flow cytometry standard FCS file types 8 6 2 Opening files 1 Click 5 to open the Open window Figure 8 8 Only E
42. button or fingertip action on touch screen Single click on circle single finger touch Double click on circle double finger touch Effect DACUK Default open circle indicates no operation Clear Closed green circle with orange rim Sample selected for measurement Orange circle indicates that the sample is activated and any alterations made to the measurement strategy e g labeling will only apply to sample positions with this designation Closed green circle Sample selected for measurement Closed blue circle Measurement in progress Closed gray circle Measurement finished Closed yellow circle Processing of sample has commenced e g sample has been labeled and incubation is underway Table 8 3 Summary of rack configurations 160 Note Colors might change if you have activated the Red Green Color blindness option under Edit gt Options Software 2 Use the right click button to select deselect single or multiple sample positions The entire rack may also be selected deselected or even cleared using the multiple sample menu button located at the top left hand corner of the dialog box Rows or columns can also be selected or deselected by clicking on the letter or number respectively User action to Effect Details select multiple sample positions Single right click of 19 Use this button to change the Select All the multiple a OL One 6 settings for all rack positions Deselect All C
43. compensation matrix This matrix organizes the detection of individual fluorescence signals by columns Column headers identify representative fluorochromes detected on the MACSQuant Instrument see Figure 4 6 The rows indicate the channels specified by the detection wavelength filter combinations Please note that in the diagonal column versus row a 1 0 is inserted as default This position within the matrix indicates the primary detection channel for that fluorescence All other positions have 0 0 inserted as defaults Instrument settings FL Channel Compensation Matrix VioBlue PI PE Cy5 5 APC Cy 1 000 CJ 2 0 000 0 000 LJ 0 000 0 000 0 000 0 000 LJ 0 000 0 000 0 000 0 000 LJ 0 000 0 000 0 000 0 000 0 000 1 000 L C 0 000 0 000 0 000 L C 0 000 0 000 T 0 000 0 000 0 000 Y 0 000 1 000 Q Figure 4 6 8x8 compensation matrix 48 When a level of spill over is detected with single stained samples values between 0 and 1 are inserted into the spill over detection channel to set the compensation adjustments As values are added into the various spill over channels the same value should be subtracted from the primary detection channel thus the entire value within the column should equal 1 0 or 100 of detected signal In Figure 4 7 spill over into the B2 PE channel is being determined on a FITC single stained cell In this example it was determined
44. detector configuration ax Choose from the list of available configurations Configuration MACS Uuant Analyzer MACS uant Analyzer MACS Ouant Analyzer 10 MACS Ouant WB Command New workspace Open Save Import FCS file Copy Print Print all Logout Optional Edit configuration Description Create a new workspace The user will be prompted to save any changes to the workspace before this action is performed Open Workspaces Instrument Settings Experiments Analysis templates and or Data files depending on the user access rights set by the administrator Click to save Workspaces Instrument settings Experiments Reagents and Analysis templates depending on user access rights set by the administrator Import data files in the FCS file compression format Copy files and templates to e g external media or from external media on the MACSQuant Instrument Print a selected area Print the entire workspace Logout of the current session Optional Only without hardware Click to choose configuration 29 Edit Edit View Mode Analysis Window Help FF fe CS WO I Undo trz Redo Ctrl Copy page ctrl E Copy plot Ctrl Shift C Delete region Del Elipse Rectangle Polygon Quadrant Interval User settings Instrument settings Chrl Alt 1 fess Rack Ctrl Ak R Reagents Options Configuration
45. file When using the software for analysis users can use the channels tab to redefine data display scales of previously acquired data files 3 4 User administration 3 4 1 Creating a new user In order to optimize the unique user management system of the MACSQuant Software it is recommended to create an individual user account for each user 33 1 Select Edit in the Windows pull down menu and User settings List of Users User Initials Group Rights Directory Public Private Password adm admin AXE C cap user adminIREAD IREAd yes Figure 3 3 The users dialog box 2 Click Add to create a new user User Settings User Name New User Initials NU Group Express Custom Administrator Access Public Private Instrument Settings Read Read amp Write Experiments Read Read amp Write Reagents Read Read amp Write Analysis Read Read amp Write Data files Read Read amp Write Password C Required _ Reset Figure 3 4 Creating a new user account 3 Enter the Name Initials and associated access settings of the new user Category Sub category Description Name Enter the user identity in this field Initials Enter the respective initials Express Check radio button to setup the account for Express mode access only Custom Check radio button to setup the account for Custom mode and Express mode access Administrator Check radio button to setup the account as an 34
46. file and clicking Open 10 The instrument will proceed to Acquisition mode 11 Following data acquisition the MACSQuant Instrument will automatically proceed to Analysis mode Custom Help Logout lolelal am Analysis Rack Single tube rack i Filename EU2009 09 29 001 mqd Sample ID jl Description Mode Analysis template CD14 Figure 8 17 Analysis mode CD14 cells isolated from PMBC using MACS MicroBeads were analyzed using the MACS Control MC CD14 Monocyte Cocktail human The 171 positive fraction is shown A total of 1 56x105 viable monocytes were enriched from the Original PBMC fraction which accounted for an enrichment rate of 69 7 The analysis template CD14 automatically generated the gating strategy and associated statistics 8 8 Reading reagents with the code reader in Express mode The 2D code reader barcode reader is used to scan reagent vials Reagent vials are automatically recognized and logged by the MACSQuantify Software WARNING Read the chapter 1 of the user manual before using the 2D Code Reader To scan reagents perform the following 1 Click the activate code reader icon MN The code reader will being blinking 2 Present the reagent vial in front of the 2D code reader Ensure the 2D code is facing the blinking code reader light The optimal reading distance is 0 5 2 5 cm from the code reader cover tilt the vial as depicte
47. fluorescence channel This overlap should be determined and corrected This is especially important when multicolor analyses are to be performed without proper compensation results may be misinterpreted Multiparameter cell analysis is the examination of multiple cellular properties by the simultaneous detection of different fluorochromes identifying these properties The configuration of the MACSQuant Instrument allows the excitation and detection of up to 8 different fluorochromes in individual detection channels Please refer to Table 1 1 to see the filter detection ranges for each fluorescence channel If the combination of fluorochromes chosen have emission spectra in overlapping wavelength ranges it is possible that a certain percentage of detectable light from one fluorochrome could be detected or spill over in an incorrect detection channel e g FITC fluorescence should be detected in the primary B1 channel but is also detected to a certain degree in the B2 PE channel with the MACSQuant Instrument See Figure 4 4 This overlap should be determined and corrected This is done by utilizing cells or antibody capture compensation beads that emit fluorescence signal from only one fluorochrome at a time allowing the user to apply compensation 45 PE spill over adjustments to the instrument settings Proper compensation is absolutely necessary for proper analysis of multiparameter cell analysis FITC PE bandpass FITC emissio
48. means electronically mechanically by photocopying microfilming recording or otherwise without the prior written consent of Miltenyi Biotec however notwithstanding the foregoing the owners of the MACSQuant Instrument may make copies solely for purposes of training personnel in the use and servicing of the unit within their business or organization Maximal care has been taken by Miltenyi Biotec in the preparation of this user manual However Miltenyi Biotec shall not be liable for any technical or editorial errors or omissions contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this document The information in this document is provided as is without warranty of any kind and is subject to change without notice autoMACS MACS the MACS logo MACSQuant MACSQuantify Vio VioBlue and VioGreen are either registered trademarks or trademarks of Miltenyi Biotec GmbH or its affiliates in Germany the United States and or other countries Ficoll and Ficoll Paque are trademarks of GE Healthcare companies BD and BD Falcon are trademarks of Becton Dickinson and Company All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only Unless otherwise specifically indicated Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use Software version 2 3 C
49. number events in the current gating strategy T Events in selected region are displayed as a percentage of the total number of acquired events Count The actual number of events occurring in the selected region is displayed Count uL The concentration of events per microliter is displayed Count mL The concentration of events per mL is displayed Table 7 1 An overview of the Properties settings for dot plots and density plots Overview of the Properties settings for histograms A variety of display settings for histograms can be modified using the Properties tb i 7 option from the drop down menu 1 Click the icon located adjacent to the histogram 2 Click Properties MWM The properties window will appear Note Refer to Table 7 2 for an overview of the various display Properties that may be modified for histograms using the MACSQuantify Software Tab Category Property Associated screenshot Description option View Data All URA r All events are displayed on the O Fixed number 1000 vj lt gt histogram Percenti le 50 v Percentile 50 25 5 2 or 1 percentile values of the total events can be displayed on the histogram This is useful when too many events have been acquired and the displayed plot histogram is saturated Fixed number A fixed number of events can be O Percentile 50 v Fixed number Ga al lt gt displayed on the histogram Numbe
50. of instrument refer to the following tables MACSQuant Analyzer Excitation Photomultipler tube PMT name wavelength 405 nm V1 VioBlue 488 nm FITC 2 PE B3 PE Cy5 PE Cy5 5 B4 PE Cy7 APC APC Cy7 488 nm FSC SSC MACSQuant Analyzer 10 Excitation Photomultipler tube PMT name wavelength 405 nm V1 VioBlue 2 VioGreen Pacific Orange 1 FITC 2 PE 3 PE Cy5 PE Cy5 5 PE Cy7 1 APC APC Cy7 488 nm FSC SSC MACSQuant VYB Excitation Photomultipler tube PMT name wavelength 405 nm VioBlue CFP VioGreen Pacific Orange 1 FITC GFP 2 PI 1 PE 2 mCherry dsRed PE Cy5 mKate Y4 PE Cy7 Filter 450 50 525 50 585 40 655 730 655LP split 730 750 LP 655 730 655LP split 730 750 LP 488 10 Filter 450 50 525 50 525 50 585 40 655 730 655LP split 730 750 LP 655 730 655LP split 730 750 LP 488 10 Filter 450 50 525 50 525 50 614 50 586 15 615 20 661 20 750 LP 488 nm FSC SSC 561 10 Table 1 1 Summary of compatible fluorochromes and respective channels After antibody labeling the needle arm of the MACSQuant Instrument draws a pre definable volume of the cell sample into the instrument Cells are either transferred directly to the flow cell for analysis or can be diverted to the MACSQuant Column for pre enrichment of magnetically labeled cells see section 6 Once in the flow cell each cell individually passes
51. oo eee Figure 3 8 Instrument is in data analysis mode Green MACSQuant in Acquisition mode calibration was successfully performed one day ago time in days since the last calibration is indicated A Acquisition mode Time Volume Rate Count 20 03 Calibration ok 1 day old ooo i ow sd _ rec _ p Figure 3 9 Instrument is in acquisition mode Yellow Cleaning and priming of MACSQuant Instrument instrument is not available for measurement cleaning in progress Cleaning MACS Quant an Time Volume Rate Count gt C ji ___00 00__ Oyl 7 0 Figure 3 10 Instrument is in the process of being cleaned Grey Instrument is being initialized instrument is not available 15 09 Initializing MACS Quant Figure 3 11 Instrument is unavailable 39 Blue MACSQuant Instrument is processing a sample Instrument is currently processing a sample 1 8 1 5 Processing rack ar Measuring sample Volume Rate Count Ceo 7 ooms fo _raieo ETT 0 23 1 414 Figure 3 12 Sample processing in progress Note Upon completion of the initialization process the MACSQuant Instrument is in the Data analysis mode until the instrument is placed in Acquisition mode Checking the instrument status using the bottle LEDs The MACSQuant Instrument is equipped with light emitting diodes LEDs which illuminate each bottle to indicate the status of the instrument in Acquisition mode Green bottl
52. or for comparing intensities of multiple samples for a single parameter overlays 1000 750 SSC A 500 250 0 250 500 750 1000 1 0e2 1 0e 1 1 0e0 1 0e1 1 0e2 1 0e3 FSC 4A CD3 PE A Figure 1 2 Left FSC SSC dot plot of PBMCs that were stained with CD3 antibodies conjugated to PE CD3 cells are depicted green Right Corresponding histogram of the CD3 cell population fluorescence intensity x axis is plotted against relative cell number y axis Density plot With traditional dot plots it may be difficult to quickly determine the intensity or frequency of acquired events on a black and white graphic A density plot is displayed in grayscale or in color each color shade provides information about the intensity of acquired events In essence the density plot is designed to represent a three dimensional plot where the number of cell events are depicted in a third dimension either by shades of grey or by using different colors oe Grapulocytes S5C A 0 50 500 750 1000 FSC A Lymphocytes Figure 1 3 Representative density plot of peripheral blood mononuclear cells The red color represents the highest density of cells followed by yellow then green and finally royal blue which represents the cells occurring at the lowest frequency Statistics Before performing statistical analysis of cell populations it is important to note that the precision and relevance of the analyzed data is dependent on the sampl
53. reevaluation of compensation values Compensation can be set using antibody capture compensation beads However remember to not change fluorescent PMT voltages after setting the compensation The single stained compensation controls must include a positive and negative population within the sample It is best to aim for a positive population frequency of gt 10 If using an antibody that detects a rare cell population it might be possible to use compensation beads a control cell line of similar autofluorescence meaning positive and negative signal remain on scale or another antibody coupled to the same fluorochrome that labels a greater frequency of cells in the sample 47 8 If using tandem fluorochromes the exact same lot from the same manufacturer must be used to set compensation adjustments In these instances compensation beads or control cell lines can be useful Please note do not mix lots of tandem fluorochromes 9 Proper compensation should not be performed using visual inspection of fluorescence intensity Statistical analysis of the median fluorescence intensity for the spill over channel must be used to set the adjustments 10 It is best to only compensate for the channels that are being used in the experimental panels 11 Using hlog scaling for data display is preferred when visualizing compensated data 4 4 8x8 Compensation matrix in MACSQuantify Software The MACSQuantify Software uses a 8x8 or 7x7
54. stipulated using this menu To assign properties to a user group 1 Click Edit and Options Regions Windows Views Figure 7 14 The users options window 2 Click the desired radio button Express Custom or Administrator 107 3 Check the Password box if you wish to set a password as a prerequisite A passport is required for this group 4 Click OK to save changes The defaults for the user setting dialog will be set EA C Read amp Write Read amp Write Analysis Read Read amp Write Data files Read Read amp Write Password V Required Reset To assign properties to user file paths 1 Click Edit Options and Files 2 3 fo Options Flee E Users Experiment Instrument Annotations Software Keyboard Timers Acquire Export Statistic FCS Backup Regions Windows Views information Files Public global cas Private user cas Logon name Public backup Logon name Private backup Path Project Date File Initials Date Figure 7 15 Changing default file paths for user groups Public To change the location of the global folder click and select an appropriate file location To make a new folder click Private To change the location of the private folder click and select an appropriate file location To make a new folder click Change the values betw
55. that 15 of the detectable FITC signal was being seen in the B2 PE channel Therefore a value of 0 15 is added to the fourth box down in the third column of the compensation matrix The 1 0 is then readjusted to 0 85 85 of detectable signal If additional spill over of the FITC signal is found in B3 PE Cy5 channel values should be added into the fifth box down in column 3 Bl 525 50 B2 585 40 B3 655 LP roomed c 726 Warelengih rirmi Figure 4 7 Spill over into the PE channel 4 5 Compensation on the MACSQuant Instrument The MACSQuant Instrument and MACSQuantify Software allow the user to set compensation adjustments in the following ways 1 Manually compensation using the 8x8 7x7 compensation matrix 2 Automated compensation using the Express program CompensationMultiColors requires MACS MiniSampler and Chill Racks 3 Automated compensation using the Express program Compensation requires single tube holder 4 Recompensation offline within MACSQuantify Software after data acquisition 49 4 5 1 Manual compensation using the 8x8 7x7 compensation matrix Materials required e Single stained controls representing all fluorochromes to be used in the experimental staining panel Ensure that there is a comparable positive and negative population for setting compensation e 12x75 mm round bottom tubes e Single tube holder 1 Ensure the MACSQuant Instrument has been properly calibrated refer to the us
56. the administrator Backup data to DVD or initiate data transfer to USB or network location 1i Activate the 2D code barcode scanner Activate touch screen keyboard Help Open help file Custom Switch to Custom mode Only available to users with custom or administrator rights Logout Click to logout from the session 9 Main instrument control Click to switch between Acquisition mode Data analysis mode or Instrument off Table 8 1 Express interface icons with brief explanation 8 2 Login to Express mode 1 Select your user name from the dropdown list and enter the appropriate password if required 2 Click login to proceed Note If the user has been registered as an Express mode user the MACSQuantify Software will automatically log into the Express mode If the user has been registered as a Custom mode user the MACSQuantify Software will automatically log into the Custom mode Note Please contact your administrator if there is no suitable user name and or the password is incorrect MACSQuant fy User admin x Password Er X con Figure 8 2 Logging in the user Express User to the MACSQuantify Software in Express mode 157 8 3 Switching to Express mode from Custom mode 1 In Custom mode click Express mode button in the top right hand of the navigation bar admin Hele a caa 0 I Il BEE 9909 Figure 8 3 Switching to Express mode from Custom mode
57. the data file As this information varies according to the size of the data file the text header may also vary in size Some non Miltenyi Biotec flow cytometry data handling software are unable to work with files which have a larger info text header It is therefore recommended to disable this function by default Compatible Export data for use with other flow cytometry analysis software for example FlowJo In actual fact data is exported as float and linear data 3 Click Apply to implement changes Click OK close the window 117 To assign default settings for backup 1 Click Edit Options Software and Backup Users i Experiment Instrument B Software Files H keyboard Timers Acquire Backup Backup Always data files Deletion O Automatic Regions F Windows Views Figure 7 26 Changing the default file backup settings 2 Use the drop down list and or radio buttons to activate deactivate a feature Files backup e always ask before performing a backup the user is prompted to verify which file types are to be backed up e always all files all files are always backed up there is no user prompt to verify this procedure e always data files only data files are backed up there is no user prompt to verify this procedure 3 Files deletion e automatic automatically overwrite or delete files during backup e ask the user is prompted to verify deletion or overwriting o
58. the pre selected plot window middle Right Double clicking on the middle plot window enlarges the plot to cover the entire analysis window 136 5 Use the icons to select a geometrical shape for gating oldal Note or Interval can be only used for Histogram analysis in order to calculate statistics for that particular region 6 A region P1 was drawn using the polygon tool to exclude unwanted debris and select for lymphocytes MiN2009 02 27_2053 021 1000 a 750 SSC A 500 250 4 le 2 1e 1 1e0 lel le2 1e3 CD4 FITC A Figure 7 41 A polygon region P1 was drawn to exclude unwanted debris and select for CD4 lymphocytes 7 A polygon region P2 was drawn using the polygon tool to exclude unwanted dead cells on the P1 population MiN2009 02 27_2053 021 P1 PIZPE Cy5 A le 2 1e 1 1e0 lel 1e2 1e3 Anti IL 17 PE A Figure 7 42 A polygon region P1 P2 was drawn to exclude unwanted dead cells 137 8 9 A rectangle region P3 was drawn using the rectangle tool to select for IL 17 CD4 viable lymphocytes MiN2009 02 27_2053 021 P1 P2 CD4 FITC A 1e2 1e3 1 1e0 lel Anti IL 17 PE A Figure 7 43 A rectangle region P1 P2 P3 was drawn to select for activated IL 17 CD4 lymphocytes The region P3 gate i e P1 P2 P3 was displayed using the axis Anti IL 17 PE versus CD154 APC MiN2009 02 27_2053 005 P1 P2 P3 f live i y MiN2009 02 27_2053 005 C3 P2 P3
59. use 7 15 6 Stop gate What is a stop gate Unlike with a live gate all data are acquired and saved by the so called Stop gate However a Stop gate used in combination with the Events option instructs the MACSQuantify Software to acquire data from the entire analysis window until a pre defined number of events are acquired within the Stop gate i e a gate or region that is defined as the stop gate SI CUVUDI US 1F_2Ud0 Uuce Annotations Autolabel Settings Custom Express Instrument setting CD14 Experiment 1 v SSC A Analysis template Cell Cycle odora cells Gate SS Evak 10000 LJ 0 250 500 750 1000 FSC A Figure 7 51 Region P1 was defined as a stop Gate When 1 000 events are acquired within region P1 data acquisition will automatically stop and all acquired data i e the entire analysis window will be saved Stop gating strategies can be saved for future use as Analysis templates 149 To perform stop gating 1 Click to open a new analysis window Choose the required plot design 2 Define the Experiment settings as required e g uptake volume sample name flow rate labeling strategy etc 3 Enter the required number of events for the associated stop gate O Evens 000 9 4 Check the Gate option and select the desired Stop gate from the drop down list Annotations Autolabel Settings Sein Express Instrument setting CD14 Experiment 1
60. when prompted In the file folder window select the files or folders to be copied Data files as well as other files e g instrument settings and templates can be copied Select copy When all files are copied a report dialog box appears You can close the box to perform other copy or deletion functions If using a USB thumb drive the option to close and eject is available Close and eject will allow safe removal of the USB device If eject is not chosen at 93 9 this time go to Tools tab and select remove external media to stop the USB device safely To remove files that have been copied the delete button in the copy dialog box can be used Folders or individual files can be deleted The fcs file format can also be deleted by this function 7 7 Viewing analysis template used during data acquisition The file format mqd is able to save analysis templates that were used when data was acquired These analysis templates can be opened and modified during data analysis 1 2 3 4 5 Right click on data file within Samples tab Select apply analysis template see Figure 7 5 Analysis template used during file acquisition will be displayed The Analysis Mode icon will be selected when analysis template is opened To modify gating strategy or display window deselect the Analysis Mode icon and modify as needed Samples Experiment Tools Channels 1 1 i live 0 0 0 Ta Open Chrl 0 fest I
61. will prompt to initiate the calibration procedure WW Scan result E i Calibration Particles detected LOT 5080414001 Would you like to proceed with calibration process Note When scanning MACS Reagents the MACSQuantify Software will prompt the user to place the vial s on the MACS Reagent Rack Note If the code reader fails to recognize the 2D code enter the information directly into the MACSQuantify Software reagent management window 5 4 Multisample processing Multisample processing is accomplished by use of the MACS MiniSampler in combination with the MACS Reagent Rack 4 and Chill Racks sample tube racks Five different kinds of sample tube racks are available Single tube rack Chill 5 Chill 15 Chill 50 and Chill 96 Rack allowing for processing of up to 96 samples in a single batch The MACS MiniSampler can be configured to perform measurements from any sample position Depending on the user s instructions samples can be labeled with fluorochromes and or measured 70 Rack type Slots Maximum Option on Corresponding MACSQuantify number MACSQuantify Rack Rack graphic of drop down list samples Single Ix5mL 1 5mL Not applicable tube rack tube Chill 5 24x5mL 6 5 mL tubes Chill 15 15x15 5 15 mL tubes 5x5 mL Chill 50 3 50 mL tubes Chill 96 96 well microtiter plate Table 5 2 Overview of the various rack types that may be used with the MACSQuant Instrument An appropriate rack sho
62. 0 250 500 750 1000 1 0e 2 1 0e 1 1 0e0 1 0e1 1 0682 1 0e3 PLA PLA Figure 1 6 Measuring quantitative changes to cellular DNA during cell cycle DNA was labeled using the fluorescent probe propidum iodide Pl A linear scale left and log5 scale right was used to plot fluorescence intensity against cell count Subtle changes to the quantity of DNA can only be visualized using a linear scale left As a general rule however changes in fluorescence intensity usually span over several orders of magnitude and therefore a logarithmic or biexponential hlog scale should be used 1 2 8 Analyzing flow cytometric data using regions or gating It is usually necessary to analyze cell subpopulations or at the very least to remove dead cells and debris from a dataset This can be achieved by defining regions of interest or gates around certain cell populations These gates are defined using geometric shapes and can be included or excluded in subsequent data analyses The following geometric shapes can be used by the MACSQuantify Software to define regions MACSQuantify DECU E Software icon z Ellipse KLa2008 07 16 001 Rectangle E 1 Ce 2 1 0e1 1 JeC 10e1 10e2 10e3 CD3 PEA oN Polygon Freehand shapes can be drawn _ low Quadrant Two parameter dot plots can be subdivided into four quadrants UL Upper left LL Lower i left LR Lower right aes m UR Upper right Intervals or markers can
63. 09 09 01_2022 001 vie Pojet D Rack Single tube rack SamplelD Description O O EON Eida ie ain Instrument Control Ei Medi Hi AE ae oO Mon C Switch into the acquistion mode Pickup and measure Liquid sensor C Mix sample Mode Standard 7 Uptake volume 100 ul Gal Sample volume 0 Lj Acquisition mode Data analysis mode Instrument off Annotations Autolabel Settings Custom Express C Instrument setting Setup 20090828 Detault C Analysis template C Gate C Events 10000 1 3 02 Data analysis mode Time m SEN _ ao wes Count 00 00 Opl O sec 0 Figure 3 7 Switching the instrument into acquisition mode 2 After the instrument has been primed and all system checks have been performed successfully the instrument status and LEDs will display green If the system status is red act in accordance with the error message The system status will prompt you if the instrument needs to be calibrated and how many days since the last calibration 3 Ifthe LEDs are red or if you suspect one of the solutions is too low you can replace the appropriate solution 4 To replace a solution place the closed bottle into the orange solution basket prior to exchanging it Remove the cap and attach the bottle closure and sensor to the new bottle 5 To empty the waste container remo
64. 3 Count af srv2009 02 04_2062 054 po County af srv2009 02 04_2062 053 neg 9 lt Count ml af srv2009 02 04_2062 053 ne af srv2009 02 04_2062 053 ne of srv2009 02 04_2062 053 ne af srv2009 02 04_2062 052 ori f srv2009 02 04_2062 052 ori of srv2009 02 04_2062 052 ori of srv2009 02 04_2062 052 ori Select the gates regions for export using the Regions box Fa For export Not for export Similarly use the Functions box to select deselect region functions for export Feature functions Export sample list Options Region functions Feature functions Features Functions af Time Mean f FSCA of SSCA af CD45 VioBlue A ff FITCA of CD19 PE A VW PLA gt Modal af PE Cy 7 A af CD20 4PC 4 af APC Cy 7 A In a similar manner select deselect Features i e Channels and Functions i e Statistics for export 4 After configuring the export options click OK 5 The file is exported to an Excel file and or the clipboard Note If the relevant Options check boxes have been selected use Windows explorer to navigate to the exported Excel file or alternatively paste the data into another windows application using the Edit Paste command 154 8 Express mode The Express mode is designed to simplify the setup running and analysis of experiments With only a few actions users with minimal flow cytometry experience can perform complex flow cytometry exper
65. 30 xla 5 vla i A amp O 30 viel 5 vial i O 30 xfa 5 v a i B a 30 xla 5 viel i a 30 vial 5 vial i c O 30 xla 5 v a G o o xla s xjaj D o o xla 5 vlaj fi D ancel Figure 5 11 Experiment definition for sample positions Al and A2 Note that reagent vial MC CD14 Monocyte Cocktail human was defined to position one of the MACS Reagent Rack Red box Default annotations yellow were used for the analysis channels 77 4 Rack positions Al and A2 are defined 5 Left click once on the rack coordinates A3 to define experiment settings for this position 1 2 3 5 000000 tal G2 T Lo KA f i a ai Mad R Ned 7 D EA r k gt a LY Mt iat Med Clear Group 6 Define the experiment settings for position A3 as shown below see es Samples Experiment Tools Channels Experiment Rack Chill 5 tube rack v C File Triall Jon vfa O Project MACS Control Cocktail CD14 l Fi Sample ID Samples 1 amp 2 Description Human PB MC Flow rate Low E Medium a High Pickup and measure Liquid sensor C Mix sample Mode Standard D E Uptake volume 50 ul yy Sample volume 200 pl B E Annotations Autolabel Settings Custom O Express Instrument setting CD14 Exptl C Analysis template C Gate CI Events 10 000 LJ E te Se Ae US a 66 Figure 5 13 The sample in rack position A3 will be analyzed in custom mode using a previously saved Instrument s
66. 70_EPC_Enrichment_and_Enumeration_Ki t_human_ aspx Materials required 2 3 4 5 6 EPC Enrichment and Enumeration Kit human autoMACS Running Buffer 12x75 mm 5 mL tubes Micro centrifuge tubes 1 5 mL 50 mL tubes Fresh human whole blood MACSQuant Instrument with installed MACSQuant Column Pre chilled Chill 5 Rack While preparing the samples for analysis turn on the MACSQuant Instrument perform a PMT calibration if necessary and open or generate appropriate instrument settings Three samples were prepared by RBC lysis and labeled with the following reagents from the EPC Enrichment and Enumeration Kit CD133 control sample 200 uL of lysed whole blood labeled with FcR Blocking Reagent and EPC Control Cocktail CD133 CD309 control sample 10 mL of lysed whole blood labeled with FcR Blocking Reagent EPC Enrichment Cocktail and EPC Control Cocktail CD309 EPC sample 10 mL of lysed whole blood labeled with FcR Blocking Reagent EPC Enrichment Cocktail and EPC Staining Cocktail All three samples were resuspended to 500 uL and transferred to a 12x75 mm tube The three samples were placed in the following positions in the Chill 5 Rack e CD133 control sample Al e CD309 control sample B1 e EPC sample Cl Program the Experiment tab in the following manner Select Chill 5 Rack 84 7 Highlight Al B1 C1 8 With all three positions highlighted enter 500 uL in the sample volume 9 Optional En
67. Analysis template Gate C Events y Data analysis mode Time Volume Rate Count 17 34 NE Le LS EE EE a 92 4 5 6 Select all files or all data files when prompted When back up is complete a dialog window appears When the back up is performed again all previously backed up mqad files will be deleted if selected Choose delete cloned files All fcs files will need to be deleted manually through the copy function see below Transferring data using copy function 1 2 3 4 5 6 7 8 If using a USB thumb drive to transfer data insert into available USB port and wait a few seconds for the instrument to be recognized Open the Copy function from the File menu Choose the copy location from the available drop down menu USB thumb drive network location etc Copy 21x Ta To x ce C 7 GB available Eject Tay ll Workspaces Directory f Am too o oo ef S 7 lt ge MOSOFTSETUP E H E 4 color sample MJ2009 07 03_2112 021 fcs B Izy B Public backup place Z C PUD PTT autocomp mqd B Analysis templates C C Colin comp mad B C comp APC MJ2009 07 03_2112 005 fcs sal C i comp PE MJ2009 07 03_2112 003 fes B Bia ahaa C C comp PerCP MJ2009 07 03 2112 004 fes B Experiments Other files 4 gt 0 files selected 0 Byte a Dette Conca _ Log files Ls If using a network location to transfer file enter password
68. CD309 APC A Top row CD133 control sample Middle row CD309 control sample Bottom row EPC sample 7 Working with data files in Custom mode 7 1 Introduction to file handling This section describes how data files can be opened saved and backed up in Custom mode Data files may be stored to and therefore opened from a Public Private or External file location Public Private aa e Public files are located on the local hard drive of the MACSQuant Instrument or personal computer and are accessible by all users e Private files are located on the local hard drive of the MACSQuant Instrument or personal computer and are only accessible by the logged in user account e External files are located on an independent file storage device which is connected to the MACSQuant Instrument or personal computer via the USB port i e amemory stick External The default window for saving and opening data files is composed of the following tabs i Qj Open i mS Figure 7 1 The default window for opening and saving various file types Note The availability of these tab options is dependant on the user profile Custom user Express user or administrator and whether data settings are being saved or opened 88 Tab option Description The Workspaces tab allow users to save user an entire workspace which is composed of instrument settings experiment and reagent definitions and an analysis template with accompanying data
69. CSQuant Analyzer 10 at is also referred to as instrument Sterile and ready to use buffer for flow cytometry cell enrichment and washing programs The tubing connector is color coded blue Sterile and ready to use solution for overnight and longterm storage of the MACSQuant Instrument The tubing connector is color coded black Sterile and ready to use solution for washing and special cleaning programs The tubing connector is color coded green Vented screw on closure with fluid uptake tubes The bottle closures contain fluid sensors and are equipped with sensor cable connectors Each bottle closure is color coded Luer to thread connector holding the MACSQuant Column Column substitute Column devoid of any paramagnetic particles This column substitute can be used when enrichments are not being performed or for long term storage and shipment The column substitute cannot be used for cell enrichment prior to analysis FITC Fluorescein isothiocyanate Fluid container Fluid sensors Fluid sensor cable 1 5 L bottle that holds the system fluids for operational use of the MACSQuant Instrument Fluid sensors monitor the fluid levels in the containers for Running Buffer Washing Solution Storage Solution and waste via electrolyte conductivity These sensors measure electrolyte conductivity and is integrated into the closures of the Running Buffer Washing Solution Storage Solution and waste fluid containers
70. CSQuantify Software Note This information is only applicable to personal computer users of the MACSQuantify Software Please refer to the MACSQuant Instrument user manual for instructions on how the instrument may be cleaned and shutdown 1 Click the Shutdown close software icon 2 Click Exit to continue closing the software Click Abort to continue working with MACSQuantify Software Exit MACSQuantify 177 9 Shutdown of the MACSQuant Instrument The chapter outlines how a manual and automatic shutdown procedure is made 9 1 Manual shutdown If no further measurements are required shut down the instrument as follows 1 Click to select data analysis mode or instrument off This automatically starts a washing protocol that lasts for approximately seven minutes which includes an incubation of the washing solution in the fluidics followed by the flushing of the washing solution and replacement with the storage solution Note Data can be analyzed using the software interface even after the instrument has been shutdown using the analysis mode 9 2 Automated shutdown An automatic shutdown procedure can be activated using the MACSQuantify Software This may be used to prevent the nonessential illumination of lasers which shortens diode lifespan or to guard against accidentally leaving the instrument on overnight or over the weekend Configuring automatic shutdown Note Configuration of the automatic shutdo
71. Entering sample ID and description text 1 Click on the Sample ID or Description text box 2 Either the appropriate alphanumeric text using the keyboard 3 The data is automatically stored in the field 8 5 3 Mode The Mode drop down box has two options e Analysis template Analysis templates simplify data analysis so that even inexperienced flow cytometry users can perform complex data analysis Analysis templates e g gating strategies can be only created by administrators or custom users For example the following gating strategies were defined by an administrator or customer Cell Cycle Analysis CD14 analysis CD14 Cell Cycle odora cells Cell Cycle Analysis 162 e Analysis The Analysis option from the Mode drop down list reveals a list of options available to perform flow cytometric cell analysis For example CD14 allows for analysis of cells separated using CD14 MicroBeads i e application of the MACS Control CD14 Monocyte Cocktail e 8 a um amp Help Custom nt d Definition Rack Chill Stuberack w Filename NS2009 04 28 001 mqd Sample ID Description Textfeld Figure 8 6 Location of the mode drop down box Note Analysis templates or Analysis options cannot be created or modified by Express users Changing an analysis template 1 Select Analysis template from the Mode drop down menu Mode Mode
72. H RCH RCE SCHR CH New calibration files have been created under K 538 V via ae lt Setup PBMC amp colors cig Setup Gepress PBMc amp colors cig Setup 70090612 PBMC 8 colors cio Setup 200906 12 bepress PBMC 8 colors ci g Vi Ti V2 Ti 378V via 458V via mja R1 Ti R2 Ti 386V via W a Red V vi mja 526 V via Ea 122 00 via 57 Calibration settings window after creation of a bank setting ap a B FEEEEEETEF Tel o x Difference in voltage between current calibration and assay specific setting 58 5 Define an experiment in Custom mode Note Refer to section 3 for an overview of the MACSQuantify Software custom mode interface Before setting up an experiment the following questions should be addressed 1 How many samples will be analyzed and what is the sample volume e Single samples are processed using the single tube sample rack e Multiple samples up to 96 are processed by using the MACS MiniSampler in combination with one of the following Chill Racks Rack type Slots Chill 5 24x5 mL Chill 15 15x15 mL 5x5 mL Chill 50 Chill 96 96 well microtiter plate e Choose the appropriate rack and configure it accordingly Samples Experiment Tools Channels Experinerit Rack Single tube rack File adma2nns 03 08 00m Piet SD Sample ID F besp SSS 2 Will autolab
73. MACSQuantify Software guide Basics of flow cytometric analysis and software guide Version EN Original instructions Miltenyi Biotec GmbH Friedrich Ebert Strake 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 0 Fax 49 2204 85197 E mail macs miltenyibiotec de www miltenyibiotec com Read the MACSQuant A WARNING Instrument user manual before using the instrument Before using the instrument read the chapter Important safety information and all other information contained in the user manual including any safety and operating instructions Pay special attention to all warnings displayed on the instrument Failure to read and follow these guidelines could lead to improper or incorrect usage and result in damage to the instrument Improper usage could also cause severe personal injury death unpredictable results instrument malfunction and premature wear to components shortening the lifetime of the instrument Such actions may void your warranty Keep the user manual and any other safety and operating instructions provided with the instrument in a safe place accessible to all users for future reference If you have a serious concern regarding the safe use of your instrument please contact your authorized Miltenyi Biotec service provider or call Miltenyi Biotec Customer Service No part of this user manual may be reproduced stored in a retrieval system transmitted published or distributed in any form or by any
74. NSTRUMENT SETTINGS 45 4 2 1 Compensation of spectral overlap 45 4 3 GENERAL GUIDELINES FOR PROPER COMPENSATION 47 4 4 8X8 COMPENSATION MATRIX INMACSQUANTIFY SOFTWARE 48 4 5 COMPENSATION ON THE MACSQUANT INSTRUMENT 49 4 5 1 Manual compensation using the 8x8 7x7 compensation matrix 50 4 5 2 Automated compensation using the Express protocol CompensationMultiColors 51 4 5 3 Automated compensation using the Express protocol Compensation 52 4 5 4 Usage of instrument bank settings 55 5 Define an experiment in Custom mode 59 5 1 SWITCHING TO EXPRESS MODE FROM CUSTOM MODE 65 5 2 PRINTING IN CUSTOM MODE 733999 9 rrr 65 5 3 REAGENT MANAGEMENT gt gt gt gt 99 9 66 5 3 1 Selecting and assigning reagents manually using the MACSQuantify Reagents dialog box 68 5 3 2 Scanning reagents with the 2D code reader 69 5 4 MULTISAMPLE PROCESSING sSoSo 45 SSS eee 70 5 4 1 Selecting a sample rack 71 5 4 2 Configuring the sample rack for an experiment 72 5
75. RATION 24 2 4 LOGIN ON TO MACSQUANTIFY SOFTWARE 26 3 Custom mode 27 3 1 CUSTOM MODE QUICK REFERENCE GUIDE 27 3 1 1 Quick guide to the top menu bar icons 27 32 OPTIONS Sme a a ae aE 32 6 TABS AASE REEE 33 3 4 USER ADMINISTRATION 33 3 4 1 Creating a NeW User 52a 33 3 5 GETTING STARTED IN THE CUSTOM MODE 36 3 5 1 Turn on the instrument 36 3 5 2 Login as administrator or custom user 36 3 5 3 Activate the touch screen keyboard on the MACSQuant Instrument 37 3 5 4 Check the fluid levels 37 3 5 5 Check the instrument status 39 4 Instrument calibration and compensation of spectral overlap 41 4 1 CALIBRATION OF THE INSTRUMENT SETTINGS 4 4 1 1 Performing a fully automated calibration 4 4 1 2 Performing manual calibration 44 4 1 3 Setting the dilution and mixing of the calibration beads prior to calibration 44 4 2 COMPENSATION OF THE I
76. There are four positions available on the MACS Reagent Rack 4 for reagent vials Reagents can be entered using the 2D code reader or manually using the Reagents window 66 Pos Category Reagent Time Titer Order P 7 RI Human PE CD45 PE human we 10 vfa nyja 5 val A R2 Calibratior MACSQuant Calibrat L 5 vja 1100 a 5 xja f J R3 Calbratior MACSQuant Calibration Beads abg Sml o v a 1 100 vf 5 xja d R4 o vlaj 100 vla E5 yal T R5 a vla m va 5 lal R6 E 0 vja 110 vja C 5 xla ma R7 0 v a 1100 vla C5 fal R8 0 la m00 v a 5 lal 52 D vja 10 1 via 5 via J Fe r Figure 5 3 The reagent rack Reagent positions 1 2 3 and 4 are clearly marked on the MACS Reagent Rack and correspond to positions R1 R2 R3 and R4 of the MACSQuant Instrument reagent management window The MACSQuant Instrument reagent management window allows users to select reagents from a drop down list and assign reagents to a position on the reagent rack The available options are described below BB Reagents emme Pos Category Reagent Time Titer Order 7 RI Human PE CD45 PE human l 10 vfa n a 5 xia J R2 Calibratior MACSQuant Calibration Be o la 1100 vf 5 la R3 o v a 100 vla C5 fa R4 o v a m00 vla E5 al R5 o vlaj 100 va E5 fa R6 0 vla 100 via C5 fa R7 o vlaj 100 vfa E5 lal R8 o v a 1 100 vla C5 fa 2 IMA 10 1 via 5 vija Apply Cancel
77. W rs data copied to DVD Ey oe Abpea ee Insert DVD into Click deleted from the DVD hain with 0 and MACSQuant Backup Aago MACSQuantify H i Software Sua J T oodd wd ked r Click Restore backup as successfully backed up copied to the DVD RW v computer Figure 7 11 Schematic of the MACSQuantify Software MACSQuant Instrument DVD backup procedure 7 12 2 To perform backup to a USB memory stick 1 Ensure that no network drive has been defined as the default location for backup files Please contact your administrator for further advice 104 2 Insert a memory stick into the MACSQuant Instrument USB port or USB porta personal computer Wait a few seconds 3 Click 4 The files will be automatically written to the USB memory stick Restoring files from a USB memory stick to a personal computer 1 Start MACSQuantify Software and login to a user account 2 Insert the memory stick into a USB port of a personal computer with MACSQuantify Software installed 3 Click File and Import External media Eg More than one external device found Please select one and press ok ow REMOVABLE D ptt cg REMOVABLE E Figure 7 12 If more than one external USB is attached you will be prompted to selected the correct external instrument 4 Use the dialog box to select the type of file for import e g Workspace Instrument settings Data files etc Note If a copy of the imported fil
78. _cb4_T_Cell_Cocktail_h mcC_cD14_Monocyte_Cocktail_h 9mc_cb19_B_Cell_Cocktail_h Peme L My Documents My Computer gt 4 My Network File name y Places Files of type FCS Files fos Figure 7 10 Importing an FCS File 3 Navigate to the file using windows tabs Highlight the file and click Open 99 4 The file will be imported To import MACSQuantify Software files from an external source perform the following steps 1 2 3 4 5 Insert the external media to the MACSQuant Instrument USB port or computer USB port Note If no external device is attached to the MACSQuant Instrument or personal computer the following error will be displayed External Click File You can copy files from external media Highlight the type of data which should be copied choose to or from and the source To _ se OSDisk C 80GB available Directory Name Size H C G Private 4 C G Public Analysis templates Instrument settings Experiments a Other files ia O files selected 0 Byte Log files eS Delete Copy Select the file type Workspaces Instrument settings Experiments Reagents Analysis templates Data files Click Open The file s will be imported 100 7 11 Exporting files MACSQuantify Software data and instrument settings can be exported to a USB memory stick To export MACSQuantify Software files to an external source perform the following steps
79. a sample position s using the left mouse button or touch screen An entire row column or table can be selected 2 Use left mouse button or touch screen to toggle between O and d b designed sample will be measured and associated Experiment definitions for this sample can also be modified using the Experiment tab e g sample name labeling strategy uptake volume etc designed sample will be measured and associated Experiment definitions can not be changed 3 Use the Experiment tab to change the Experiment Flow rate and Pickup and 4 5 measure options as required Note To reiterate Only sample positions with status can be programmed using the Experiment tab Note For more information about defining an experiment refer to section To close the popup rack window click the Rack icon Before starting the run check that Experiment definitions are correctly assigned to each rack coordinate and that each sample is correctly positioned on the Chill Rack Sufficient quantities of reagents and buffers are provided Ensure that the waste bottle is empty The reagents have imported and assigned to a position on the MACS Reagent Rack see section 5 3 The instrument is correctly calibrated and compensated see section 4 75 5 5 Defining an experiment with multisample processing A work through example 5 5 1 Background In the following example CD14 cell
80. adi D lt add cack BET caki cmd E cr 3 Check the desired reagent position R1 R2 R3 R4 S1 or S2 This must correspond to the correct position on the reagent rack 4 Highlight a category that corresponds to the desired reagent using the Category drop down list 5 Highlight the desired reagent from the Reagent drop down list 6 Modify the incubation time Time label sample titer Titer and order if required 7 Click Apply to apply changes and close the window 5 3 2 Scanning reagents with the 2D code reader To enter regents manually into the reagents database The 2D code reader barcode reader is used to scan reagent vials Reagent vials are automatically recognized and logged by the MACSQuantify Software WARNING Read the chapter 1 of the user manual before using the 2D Code Reader To scan reagents perform the following 1 Click the activate code reader icon The code reader will being blinking 69 2 Present the reagent vial in front of the 2D code reader Ensure the 2D code is facing the blinking code reader light The optimal reading distance is 0 5 2 5 cm from the code reader cover tilt the vial Figure 5 5 Scanning a reagent using the MACSQuant Instrument 2D code reader 3 Scanned reagents are reported a MACSQuantify Software dialog box 4 Place the reagent onto the corresponding position on the reagent rack Note When scanning MACSQuant Calibration Beads the instrument
81. ain software functionalities So with this possibility the user can setup their analysis and acquisition in an convenient way It is possible to change settings for analysis e g like color of regions export files into FCS and also for the acquisition of data like standard annotations file names standard uptake volume and timers for standby and shutdown Files Users Experiment Instrument Format Custom Annotations Software Manon Version FCS 3 1 x Timers Acquire Export Format Statistic Best fit 16 bit Float Backup Options Regions ae a E Windows unear aata Add ext info I Compatible Views FCS p i Figure 3 2 Options dialog box under Edit Menu Section to define FCS file export requirements is highlighted 3 3 Tabs MACSQuantify Software organizes some software functionality into TABS located on the left hand side of the software screen These tabs are Samples Experiment Tools and Channels Samples tab displays list of sample files available for analysis with MACSQuantify Software Experiment tab define experimental parameters for sample acquisition When using the software for analysis users can use the experiment tab to redefine annotations of previously acquired data files Tools tab find access to MACSQuant Live Support and other functions such as calibration Channels tab view optical channel settings of a specified data file or instrument settings
82. ake needle will dilute the calibration beads to a total volume of 0 5 mL For the calibration process 100 uL will be taken up and injected into the sample port During calibration the gain and trigger for each respective channel will be automatically adjusted The MACSQuant Calibration Beads consist of two sizes of beads 2 um unstained beads and 3 um beads stained with fluorochromes to emit fluorescence in all 7 or 8 channels For more information about the MACSQuant Calibration Beads please review the product data sheet available at www miltenyibiotec com File Edit View Mode Analysis Window Help admin on MQ2209 Eesevansensssssssssssssosoe Experiment get Eia 00 00 T e Aioni File adm2011 07 21_2209 002 VJ 7 Bango T 33 55 T Rack Single tube rack Project Sample ID Description Flow rate Low Medium High Pickup and measure Liquid sensor Mix sample Mode Standard Uptake volume ca Sample volume 200 G Annotations Autolabel Settings Custom Express Instrument setting Analysis template Gate Events 10 000 CI Se Washing system AD Processing rack processing sample 00 13 Figure 4 2 Calibration is underway When the process is successfully completed the MACSQuant Instrument Status bar should report MACSQuant ready Calibration OK These settings will be aut
83. al computer Note If your copy of MACSQuantify Software is not registered please contact Miltenyi Biotec to obtain a registration code Visit www macsquant com for more information or contact your local sales representatives 23 1 Click Register on the login box MACS Quantity Version 2 0 Serial No PE Version Registered for Jon Barbour Expires in 36 days User Customer User pasni OOO Figure 2 1 Click Register to register your name and registration code The registration code is obtained from Miltenyi Biotec 2 Enter your name and registration code as it exactly appears on the document provided by Miltenyi Biotec Software registration Use following information on http w miltenyibiotec con to register your copy of MACS Quantify Software version 1 1 0910 A Activation key 4F5E 47771 Enter your name and registration key 2 3 MACSQuantify Software registration 1 Launch MACSQuantify Software by clicking on the shortcut icon 2 Click Register see Figure 2 3 24 Note Note the software version and Activation key see figure Note Figure 2 4 3 Close software 4 Complete the registration form on the supplied USB memory stick 5 E mail the completed form to mgsupport miltenyibiotec de 6 Upon receipt of a registration number from MACSQuant Support run the MACSQuantify Software click Register and enter your user name and registration number 7 Click OK to c
84. alist Please contact your Miltenyi Biotec advisor for further information Compensation matrix The values from the compensation matrix will be displayed as a table Instrument settings The current instrument settings for each channel PMT voltages and scales will be displayed asa table Experiment info from sample Experimental settings such as uptake and sample volumes sample ID and description will be displayed as a table If a MACSQuantify Script has been used text tab it possible to add or remove regions populations by using the population check box In the following example all four regions P1 P2 P3 P4 of a gating strategy were checked and therefore displayed st 2008 12 10_2063 015 pos F Population 1 siv2008 12 10_2063 015 pos P1 m Legend v Population 2 st 2008 12 10_2063 015 pos P1 P2 m Region Population Count F Population 3 s1v2008 12 10_2063 015 pos P1 P2 P3 m P1 Debris exclusion 13552 2 Viable leukocytes 11178 Z Population 4 srv2008 12 10_2063 015 pos P3 P4 a ei ae Monocytes Table 7 4 An overview of the Properties settings for text boxes 7 15 Working with regions or gates For background information concerning drawing regions with MACSQuantify Software refer to section 1 2 8 7 15 1 Drawing regions Note In the following example the IL 17 Secretion Assay Cell Enrichment and Detection Kit
85. am will use the current instrument settings from the MACSQuant Instrument to perform the compensation If the current instrument settings on the instrument are different than the default from that day an informational dialog box will appear stating that the live settings are not the default set after calibration The user then has the opportunity to choose the default setting from that day or to continue with the current settings 12 When prompted draw a region around the scatter population that defines the cells or beads used for compensation 13 When the compensation is complete a dialog box will ask to save the instrument settings 14 Choose either a Public or Private saving option and name the instrument settings Recompensation offline within MACSQuantify Software after data acquisition Recompensation can be performed on any file acquired on the MACSQuant Instrument using MACSQuantify Software To start recompensation ensure that the instrument settings used with the file of interest are loaded into the software 53 2 3 4 5 6 7 8 9 Choose the file from the sample list Right click on the file and select Apply instrument settings see Figure 4 8 Open a display template Plot3 by selecting the ea icon and add the file to the template In the left dot plot set up to visualize FSC on the x axis and SSC on the y axis In the right plot window change the plot type to histogram b
86. analysis To open saved data files 1 Click File and Open or click the icon a A 3 Choose the desired data file s from a Public Private or External source 2 Click the Data files tab Edit View Mode Analysis Window Help xdd HH SEE a e O E admin Epress IP rE rr 0 E toma File Hala AUN Sami t Tools Channels ampl Statistic J Cells live 0 0 0 i 100 14887 stv20l 0_2063 014 neg 100 18222 0_2063 013 ori 100 22130 Figure 7 47 Three MACSQuantify Software data files were opened from a public shared location Applying analysis templates to post acquired data It is possible to apply or view an analysis template that was used in a previous experiment i e the gating strategy and corresponding plots that are associated with acquired data files can be reapplied and viewed To apply an analysis template associated with a data file 1 Open the desired data file s 2 Click on the Sample tab menu 143 3 Right click on the data file and select Apply analysis template Samples Experiment Tools Channels live 0 0 0 T0z2009 01 23 lm Open Ctrl O T022009 01 23 Export sample list 4 The data file and corresponding analysis template will be loaded in analysis mode Al Experiment Tools E srv2008 12 10_2063 014 neg 100 0 E srv2008 12 10_2063 013 0r1 100 0 Figure 7 48 Application of an
87. analysis template for the file Exavzaribmcsuii The template shows a gating strategy for analysis of CD14 monocytes 144 5 To View analyzed results of the previously defined analysis right click on the data file and select View with analysis lt name of analysis gt Pere ee Pe z eeu a ug lam Open Ctri O I admin F ma E E Sa Lad Dl Oe am ee S me ze eed Mode _ _ Samples Experiment Tools fits Import Fs fie f Export sample Remove Recompensate o _ gt s1v2008 12 10_2063 014 neg 100 0 18222 s1v2008 12 10_2063 013 0n 100 0 22130 Apply instrument settings 1 0e 21 0e 11 0 01 0e11 0e2 1 023 VioBlue A Apply analysis template s1v2008 12 10_2063 015 pos 1 0e 21 0e 11 0e01 0e11 0e21 0e3 PE A stv2008 12 10_2063 015 pos 108 12 10_ 015 pos Results pos 16 log ise Freq 2 Conc 1 ml Viable leukocytes vere 82 9 2 24E 005 Viable CD14 monocytes vere 69 7 1 56E 005 1 0e 21 0e 11 0e0 1 0e11 0e21 0e3 VioBlue A Figure 7 49 Viewing results of a CD14 analysis CD14 monocytes were analyzed using an appropriate experiment design and gating strategy using the MACSQuant Instrument and MACSQuantify Software Applying previously saved instrument settings PMT voltage and compensation settings It is possible to apply instrument settings associated with a data file To apply a previously saved instrument setting that is assoc
88. and click on the Continue button 10 At the end of the process enter the desired name for the instrument setting file in the Save window and click the Save button 4 5 3 Automated compensation using the Express protocol Compensation Materials required e Single stained controls representing all fluorochromes to be used in the experimental staining panel combined into one tube Ensure that there is a comparable positive and negative population for setting compensation e 12x75 mm round bottom tubes e Single tube holder 1 Ensure the MACSQuant Instrument has been properly calibrated refer to the user manual 52 2 3 4 5 6 7 8 9 Adjust all PMT voltages for the cell sample e g FSCvSSC From the Edit menu choose Calibration Under the default tab designate the channels that require compensation by choosing the proper lab from the drop down menu No No compensation required Yes Compensate spill over out of this channel but no fluorochrome is present in the compensation sample Yes P compensate spill over out of this channel and compensate this fluorochromes signal from other channels In the Experiment tab choose single tube holder Under the Settings tab click the Express radio button Select Setup from the Type drop down menu Select Compensation from the Mode drop down menu Optional Enter sample ID project folder uptake volume etc 10 Start acquisition 11 The compensation progr
89. atistic are different Refer to the following sections for an overview of the settings available for each chart type 1 Click Apply to apply changes 2 Click Ok to apply changes and close the Properties window Overview of the Properties settings for dot plots and density plots A variety of display settings for dot plots histograms and density plots can be modified using the Properties option from the drop down menu 1 Click the icon located adjacent to the dot plot histogram or density plot 2 Click Properties 225 The properties window will appear Note Refer to Table 7 1 for an overview of the various display Properties that may be modified for dot plots and density plots using the MACSQuantify Software 129 Tab option View Region functions Category Property Data Axes Options All Percentile Fixed number X axis Y axis All This None Regions Functions Associated screenshot All Percentile 50 v O Fixed number 1000 v a lt gt Percentile 50 v O Fixed number Data O All O Percentile 50 v Fixed number 100d via lt gt As acquired OOOO O O Y axi Axes X ax act v Y axis vj Dptions Regions All v Options Regione E Options Regions v View Region functions Regions D srv2008 1 2 10_2063 013 ori sf srv2008 12 10_2063 013 af srv2008 12 10_2063 013 ori vf stv Y sv 2008
90. atter plotted against forward scatter of human peripheral blood mononuclear cells PBMCs Three distinct cells populations can be identified according to their light scattering properties granulocytes monocytes and lymphocytes Scatter scales are usually plotted using a linear scale In fluorescence dot plots the x and y scales are used to plot fluorescence intensity and since fluorescence intensity can vary by several orders of magnitude between cells a logarithmic scale is usually employed e g fluorescence intensity spanning five decades for more information about choosing an appropriate scale refer the section Scaling flow cytometry data below Dot plots are ideal for displaying relatively small numbers of events where discrete cell populations can be easily identified e g see Figure 1 1 They also provide some indication regarding the relative density of cell events i e with more events the dots accumulate forming a darker dot plot with more contrast However for a more accurate reflection of the relative density of cell events a density plot should be employed Histogram Histograms are used to plot the intensity of a single parameter x axis against the frequency of that parameter y axis i e the x axis represents scatter or fluorescence intensity and the y axis axis represents the number of events Since histogram charts can display only one dimension they should be employed for well resolved homogeneous populations
91. be displayed in a text box Refer to section 1 2 7 for an explanation of these formats Note For information about file handling in Custom mode e g opening and saving data files refer to section 7 Custom mode users can change the properties of a dot plot density plot histogram and statistic or text box as follows 1 Click on the icon located beside the dot plot histogram or text table A popup menu will appear 127 Dot plot Density plok Hiskougr an Statistic a pace io Lt Ne ie Ir Multilayer mode Properties Figure 7 36 i Popup menu 2 Click Properties MWM The properties window will appear E Properties View Region functions Data All Percentile BU Fixed number 1000 v a gt Sxes m aMis Ag acquired wt Y aiz Ags acquired Options Regions Figure 7 37 Properties window 128 3 Select the desired property and modify accordingly For example to change the axes of a dot plot use the Axes drop down menu E Properties View Region functions Data All Percentile E x Fixed number 1000 vla lt gt Aves 2 aMis As acquired Y asis As acquired Options Regions Figure 7 38 Changing the axis properties of a dot plot Note The properties window for Dot plots and Density plots is identical The properties window for Histogram Text and St
92. be drawn on histograms to calculate statistics for designated regions Table 1 2 Geometric shapes that can be used to draw gates or regions of interest using the MACSQuantify Software Complex or boolean gating Several regions can be defined to form a boolean argument or a gating strategy which only displays highly specific cell populations for example cell populations with a defined set of scatter properties and or a specific cell surface marker phenotype as identified by fluorescence labeling using fluorochrome conjugated monoclonal antibodies Gates or regions created using MACSQuantify Software are identified by the letter P where P is derived from Population An example of a complex gating strategy is shown by Figure 1 7 Sensitive rare cell analysis of CD34 cells was performed using the MACSQuant Instrument In order to visualize pre enriched human CD34 cells from a peripheral blood mononuclear cell PBMC preparation a suitable gating strategy was devised Figure 1 7 A to E Cells were autolabeled with PE conjugated anti human CD34 antibodies to detect for CD34 cells Propidium iodide solution was used to exclude dead cells from flow cytometric analysis An explanation of the gating strategy is given in the accompanying figure legend 1 30_2062 008 4P1 1 0e3 T j 1 0e2 lt Ww S 1 0e1 E eee ty 5 pi e as 3 1 0e0 been named Eo CD34 target cells Da
93. beling strategy may be applied 169 3 4 5 Select the Analysis Mode and MC_CD14_h from the lower drop down list Figure 8 13 CD14 MACS Control Cocktail analysis is applied to rack positions Al and A2 Use the Sample ID and Description fields to enter relevant sample information cample lO CO_14 Cocktail Description PMBC Preparation Figure 8 14 The above information is now associated with sample positions Al and A2 Select position A3 Figure 8 15 Position A3 is selected In doing so positions Al and A2 are automatically deselected 6 Select the Analysis Mode and MC_CD4_h from the lower drop down list 7 Use the Sample ID and Description fields to enter relevant sample information 8 Ensure that e The samples are correctly positioned on the reagent rack and that the MACSQuant Instrument is provided with adequate buffer e The waste bottle is empty e The instrument is correctly calibrated and compensated 170 9 Click Start Measurement CJ to start analysis mq Express User e Bla l mm iin custom a D Definition Rack Filename EU2009 09 28 001 mqd Sample Desorption Mode Figure 8 16 The experiment has been defined By clicking Start Measurement the instrument will change to Acquisition mode Note By saving the Experiment definition see step 7 above the user can reapply the definition by clicking selecting the appropriate
94. ch the number of samples for compensation 51 4 Click on the Group button at the bottom of the window Each of the selected rack positions should now labeled with the number 1 5 Choose Express from the Settings tab in the Experiment section 6 Select Setup in the upper drop down list and CompensationMultiColors from the lower list 7 To set the fluorochrome for each rack position highlight the respective circle in the rack window Only one circle must be highlighted at a time In the Sample ID field a drop down list will be available where the representative fluorochrome can be chosen Note Each fluorochrome name in the drop down list represents the respective detection channel For example PerCP represents the channel B3 When using another B3 compatible fluorochrome like PE Cy 5 PerCP must be selected from the list The position blank is a sample with unstained cells used as a reference for negative cells The sample PI is necessary when propidium iodide PI will be used for dead cell exclusion It is used for compensating all fluorescence channels against the channel B3 This ensures that there is no fluorescence spillover into the PI staining The sample should be unstained cells The addition of Pl is not necessary 8 Place samples in proper rack positions and click the start measurement icon 9 After prompting by the software draw a region around the cells of interest e g lymphocytes in the scatter plot
95. chnology before fluorescence cell analysis is particularly valuable when target cells occur at extremely low numbers such as stem cells or antigen specific T cells 1 4 Description of the MACSQuant Analyzer MACSQuant Analyzer 10 and MACSQuant VYB The MACSQuant instruments are state of the art benchtop cell analyzers for highly sensitive multicolor flow cytometry Three lasers combined with powerful MACSQuantify Software make for a fast and simple analysis of cells The MACSQuant Analyzer is equipped with 405 nm 488 nm and 635 nm lasers two scatter FSC SSC and seven fluorescence channels The MACSQuant Analyzer 10 is equipped with an additional fluorescence channel to provide users with eight possible fluorescence channels Thus the MACSQuant Analyzer and MACSQuant Analyzer 10 are ideal for MACS Control applications optimized evaluation of MACS Cell Separations as well as standard immunofluorescence analyses The MACSQuant VYB is also a 10 parameter instrument with two scatter and eight fluorescence channels but has been equipped with 405 nm 488 nm and 561 nm lasers for enhanced fluorescent protein analysis All MACSQuant instruments include the MACS Cell Enrichment Unit that is crucial for 20 the reliable detection and analysis of rare cells Further operation of the MACS Cell Enrichment Unit in the instruments is directly controlled by the MACSQuant Analysis Software to enable the fully automated processing and acquisition of sam
96. created within gates in order to identify sub populations of cells SMiN 2009 02 27 2053 005 100 0 fedaa4 Prerrere errr rerrrrreriririr iii ritreriereirriretirieiiiiriiiiiiii iii ig ch PI 62 6 143622 il c P2 60 93 139674 LIPS 2 4 46916 Regions having the same hierachy It is also possible to create two different regions from one parental gate Regions drawn in a single plot have the same strategy In the example below regions P3 and P4 bottom left were both defined within region P2 top right and therefore have the same hierarchy 2008 01 23 017 2001 25 001 FMP J q meng T amen ne T Meo Snes CD3 PEA NOT gates So called NOT gates are used to eliminate a cell population from analysis To create a NOT gate 1 Draw a region or gate around the population to be excluded from analysis MiN2009 02 27_2053 005 P1 P1 P2 P3 i 14 38 T 1 0e 2 1 1 0e0 1 0e1 1 0e2 1 0e3 Figure 7 46 Region P3 was drawn using the rectangle drawing tool 140 2 Inthe Samples menu right click on the region of interest i e in this case P3 Experiment Channels live 0 0 MiN2009 02 27_2053 005 100 0 229334 Gc Pl 62 6 143622 GS P2 60 9 139674 ELERAN 32973 3 Select Region properties from the drop down list 4 Check the box Not This region is now excluded from analysis Optional Select a color and or name the NOT gate as desired Region properties Name Excluded from analysis Not
97. d Experiments m EE MC_CD3_Pan T_Cell_Cocktail_h MC CD19 B Cell Cocktail_h O MC_CD14_Monocpte_Cocktailh Analysis templates a MC_CD_8_T_CellCocktai_h Gay EPC Data from R amp D H C Data MACS Quant Training Data for task J O BL Figure 7 2 Open dialog Navigate to data files or other file types to open in the MACSQuantify Software 90 7 4 Adding data files 1 Data files located within the cap folder structure as detailed in section 7 2 located on a network folder hard drive location USB memory stick etc can be added to the MACSQuantify Software 2 Right click within Sample tab and select Add see Figure 7 3 3 Navigate to data files to be uploaded samples Experiment Tools Channels Sample Statistic Cells iiye 00 0 r F Open Chrl 0 Import ECS file Export sample list Figure 7 3 Adding data files from any location by navigating Windows file structure 7 5 Importing FCS files 1 FCS files from other flow cytometry software can be imported and analyzed in MACSQuantify Software 2 Right click with the Samples tab and select Import FCS file see Figure 7 4 3 If scales need to be transformed refer to section 7 14 3 for information how to transform scales samples Experiment Tools Channels Statistic Hlive 00 0 om Open Ctrl al add Import FES file Export sample list Figure 7 4 Importation of FCS files
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99. d further analysis using flow cytometry Flow cytometers detect cells according to two basic parameters light scatter and fluorescence Cell size and granularity are inherent characteristics of a cell and vary from one cell type to another these properties are measured using the forward scatter FSC and side scatter SSC channels respectively However with the exception of transient or stable expression of fluorescence proteins relatively bright fluorescence that occurs above background auto fluorescence requires cells to be stained with fluorescence dyes This is normally achieved through the use of antibodies that target a specific protein or other biochemical antigens expressed on or within cells These antibodies are either directly conjugated to a fluorochrome or can themselves be stained in a secondary step by a fluorochrome conjugated secondary antibody indirect staining Only cells expressing the particular target antigen will be fluorescence labeled Alternatively cellular signaling or protein expression profiles can be analyzed using transient or stably transfected cells that express specific proteins fused with fluorescent proteins After excitation by a laser light emitted from fluorescence dyes can be detected in defined wavelength ranges This differs from one fluorochrome to another The use of different light filters in the flow cytometer permits the simultaneous use of multiple fluorescence dyes and thus the detection
100. d in Figure 8 18 Figure 8 18 Scanning a reagent using the MACSQuant Instrument 2D code reader 3 Scanned reagents are reported by a MACSQuantify Software dialog box Note When scanning MACSQuant Calibration Beads the instrument will prompt to initiate the calibration procedure W scan result EJ sp Calibration Particles detected LOT 5080414001 2 Would you like to proceed with calibration process Note When scanning MACS Reagents the MACSQuantify Software will prompt the user to place the vial s on the MACS Reagent Rack Note Contact your administrator if the code reader fails to recognize a 172 8 9 Printing in Express mode The MACSQuantify Software uses installed windows printer drivers to print active workspaces Note The HP Universal Print driver has been installed on the MACSQuant Instrument and has been tested with the following printers Hp Laserjet P2055d P3005n CP1515n PC2025n Hp Officejet Pro 8000 Note For a complete list of printers compatible with the HP Universal Print driver please visit www hp com go upd Please note the only the above mentioned printers have been tested with the MACSQuant Instrument Note It is also possible to print to a network printer Please contact your MACSQuant Instrument administrator or Miltenyi Biotec Technical Support for more information To print active workspaces 1 Open the desired analysis window 2 a 3 Select the desired printer Click
101. e Volumetric Measurements allow for absolute cell counting 2 Ensure that reagents samples and buffers are correctly positioned Check that the waste bottle is empty Note It is assumed that the instrument hardware and settings have been correctly calibrated The instrument should have been correctly compensated 3 Click Start Measurement gt to start acquisition 63 Following Table 5 1 shows an overview of the above described procedure for defining an experiment Selected setting Description ce Chill 5 Rack A Chill Rack will be used for this a il analysis Samples Experiment Tools Channels ENON Te m File name adm2009 Filename is automatically Fie adm2011 07 21_2203 002 A 05 12 001 generated To enter a name Fis manually uncheck the associated ERT box to the right Flow rate ei ed Sample ID and The Sample ID and Description Liquid sensor Z Mix sample Description are text fields entered by the Mode Standard v user Uptake volume 100 pl EJ ee ee Flow rate Medium flow rate was selected Custom O Express 50 uL min E Instrument setting S etup 20110721 Default E Analysis template unnamed Mix sample checked The samples will be premixed by ae activated the uptake needle Custom mode Custom mode is enabled A previously saved Instrument setting and Analysis template were selected for the analysis If the instrument settings option is not checked the last successful calibration wil
102. e MACS Enrichment Unit and the MACSQuant Column allow the possibility to reduce the number of cells analyzed to characterize the rare cell population of interest This is particularly useful for the analysis of cells present in low abundance such as stem cells dendritic cell subsets or natural killer cell subsets MACS Technology MACS Cell Separation the magnetic separation of defined cell populations using MACS Technology is widely regarded as the gold standard in cell separation MACS Technology is based on the use of MACS MicroBeads MACS Columns and MACS Separators strong permanent magnets MACS Technology can be used for the targeted cell enrichment or depletion of cell types or populations through their expression of particular surface antigens It is this technology that provides the means for the pre enrichment of rare cells for subsequent flow cytometry analysis with the MACSQuant Instrument Pre enrichment with MACS MicroBeads are a perfect complement to flow cytometry These 50 nm sized magnetic particles directly conjugated to highly specific monoclonal antibodies consist of the following properties No influence on light scatter properties of cells labeled cells can be used directly with flow cytometry Colloidal particles with a high surface to volume ratio fast binding kinetics even at low temperatures to maintain cell integrity Biodegradable and non toxic no need to remove before downstream applications as the Mic
103. e already exists on the personal computer the following dialog box will appear Overwrite 2 File C capfuser admin device Cell cycle analysis cfg _ exists Overwrite To overwrite a single file click Yes To overwrite all files for import click All No aborts the procedure Note The imported files are copied to MACSQuantify Software It is necessary to delete files off the memory stick using windows explorer Note It is of course also possible to simply move files from the memory stick to a personal computer using windows explorer 105 7 12 3 To perform backup to network drive 1 Please contact your administrator if a network drive has not been configured for backup Note If a network drive is not configured the MACSQuantify Software will search for USB and DVD backup media instead 2 Click 3 The files will be automatically written to the network drive 7 12 4 Configuring data backup settings administrators only 1 Click Edit Options Software and Backup Options Users Experiment E Instrument Software Files Keyboard Timers Acquire Chaletion Automatic Ask Export H Regions Windows Views Backup Backup Always data files Figure 7 13 Changing the default file backup settings 2 Use the drop down list and or radio buttons to activate deactivate a feature Files backup e always ask before performing a backup the user is prompted to verify which file type
104. e are logarithmic whereas the lower values are linear 20059 01 30_2062 008 a 1 30_2062 008 P2 i B00 0 400 300 200 100 0 0 250 1000 fol 500 i a n Aa m p ee 5 Pe ers a 250 ee ae aoe ened ae oe ee ee a ee ERS solo pest eL coe Ao mas was E TVAE p TN j 250 1000 00 fal fou 1000 S00 FSC A Linear 1 30_2062 0084 P2 C i 1 0e 21 0e 11 0e0 1 021 1 0e2 1 0e3 PE A Cell count Logs Cell count Cell count PE A Linear 1 30_2062 008 P2 1 0e1 1 0e2 1 0e3 PE A Hlog Figure 1 5 Comparing log and linear scales when displaying fluorescence and non fluorescence events A Dot plot showing forward scatter versus side scatter using linear scales B Histogram showing fluorescence intensity linear scale versus cell count C Histogram showing fluorescence intensity log5 scale versus cell count D Histogram showing fluorescence intensity hlog scale versus cell count There are occasions when the difference between fluorescence values in a dataset are relatively small for example when using fluorescent probes to measure quantitative changes in cellular DNA during cell cycle A linear scale must therefore be used in order to visualize these subtle changes see Figure 1 6 cell cycle O01 P1 cell cycle 0014P2 a i 3500 i 900 3000 re 2500 600 500 2000 400 1500 300 1000 200 100 500 0D
105. e light The instrument is ready to measure liquid levels are sufficient and the instrument is primed Note Please note that the lasers can take up to 30 minutes to warm up after performing the initial instrument priming Purple bottle light The instrument is measuring liquid levels are sufficient Blue light is indicative of normal instrument function during sample processing or that the instrument is busy Red bottle light Liquid level error general instrument error Red light indicates that the liquid levels are too low in a particular bottle or that the waste needs to be removed The bottle with the blinking red light will indicate which bottle needs to be tended to Additionally a message on the system status in the lower left corner will refill that a bottle change is needed A bottle can be replaced even during a measurement although replacing the waste is best when the instrument is not processing Yellow bottle light Sensor error Please ensure that the sensor is correctly attached to the bottle Note If no LED is illuminating the fluid containers then the instrument is in the Data Analysis mode and the lasers are not on 40 4 Instrument calibration and compensation of spectral overlap For optimal operation of the MACSQuant Instrument it is recommended to calibrate the instrument settings to calibrate the photomultiplier tubes PMTs of the instrument If multicolor analysis is also planned compensation is also rec
106. e refer to the user manual for more information on the components of the hardware monitor like fluidics components sample uptake or optical bench 182 13 Technical Support Miltenyi Biotec offers a full range of customer Technical Support options for your MACSQuant Instrument For support and technical questions or if you think your MACSQuant Instrument is malfunctioning please contact your local Miltenyi Biotec representative or Miltenyi Biotec s Technical Support team Germany Austria Switzerland Miltenyi Biotec GmbH Friedrich Ebert Str 68 51429 Bergisch Gladbach Germany Phone 49 2204 8306 830 Fax 49 2204 8306 89 macsTec miltenyibiotec de USA Canada Miltenyi Biotec Inc 2303 Lindbergh Street Auburn CA 95602 USA Phone 1 530 888 8871 Fax 1 530 888 8925 Toll free 800 FOR MACS Toll free 1 800 367 6227 macs miltenyibiotec com Australia Miltenyi Biotec Australia Pty Ltd Unit 16A 2 Eden Park Drive North Ryde NSW 2113 Australia Phone 61 02 8877 7400 Fax 61 02 9889 5044 macs miltenyibiotec com au Benelux Miltenyi Biotec B V Schipholweg 68H 2316 XE Leiden Netherlands Customer service Belgium Phone 0800 94016 Fax 0800 99626 macs miltenyibiotec nl Customer service Luxembourg Phone 800 24971 Fax 0800 24984 Customer service The Netherlands Phone 800 4020120 Fax 0800 4020100 China Miltenyi Biotec GmbH Shanghai Office Fareast International Plaza A Rm 2301 No 319 Xianxia R
107. e size and choice of statistic respectively The MACSQuantify Software can display a summary table of statistical attributes namely count percentages arithmetic mean coefficient of variation CV minimum maximum medium median and modal statistics In addition since the MACSQuant Instrument performs volumetric cell enumeration the actual cell count can also be displayed with each measurement Text The MACSQuantify Software Text option is a text box that may be used to enter alphanumeric characters that may be used for example to document details about the experiment the gating strategy or specific dot plot This option also allows the user to display several other experimental parameters via the drop down menu such as experimental information from the sample See Figure 1 4 E Properties Text Overlay Script Text C v Edit text via Properties Figure 1 4 The text box can be used to enter additional information or to display predefined parameters located in the drop down menu found in the text properties Scaling flow cytometry data using MACSQuantify Software Standard dot plots can be displayed in linear logarithmic or hyperlog hlog scales depending on the type of data being shown Plots which show side scatter and forward scatter typically use linear scales This is often not possible when fluorescence labeled and non fluorescence labeled cell populations are being analyzed as the difference in fluorescence sig
108. e the capabilities and handling of the instruments The MACSQuant Analyzer and MACSQuant Analyzer 10 have been designed for optimized analysis with fluorescently conjugated antibodies while the MACSQuant VYB is designed for optimal analysis of fluorescent protein expression but with any of the instruments there are many additional built in features The instruments were designed with automation in mind and for use with MACS Control reagents combinations of fluorescence antibody conjugates supplied in an optimized format for the rapid flow cytometric analysis of cell separations performed with MACS Technology The automated uptake of samples using the needle arm permits measurement of a pre defined sample volume which in turn permits an absolute quantitation of cells in a sample According to their fluorescence labeling different cell populations can therefore also be quantified A brief summary of the main design features of the MACSQuant Instruments is given below 1 2 1 Fluorescence cell analysis First and foremost the MACSQuant Instruments are flow cytometers comprised of nine or ten optical channels for the measurement of fluorescence signals and the relative size and relative granularity of cells In conjunction with the MACS MiniSampler the automated analysis of multiple samples can be performed with ease The MACSQuantify Software performs all common functions for the presentation and Statistical analysis of collected data Data can be pres
109. e user manual chapter Warnings and precautions before operation of the instrument When processing infectious radioactive poisonous or any other hazardous liquids always abide by the necessary safety precautions 3 1 Custom mode quick reference guide The quick reference guide provides and overview to the icons and layout of the MACSQuantify Software in Custom mode 3 1 1 Quick guide to the top menu bar icons File Edit view Mode Analysis Window Help els S olla eS S S alla Samples Experiment Tools Channels admin EBBEDE Experiment Rack Single tube rack F File adm2009 03 01_2022 004 Project o Sample ID Description Flow rate Low Medium High Pickup and measure Liquid sensor C Mix sample Mode Standard i Uptake volume 150 pl Sample volume 200W E Autolabel Settings Annotations FLI VioBlue FL5 PE Cy7 FL2 FITC FL6 APC FL3 PE FL APC Cy Fla PEDS 1 2 92 Acquisition mode ear Time a eae Calibration ok Clear TE Figure 3 1 Screen shot of the custom mode main screen using the admin account 27 Description Description Folder icon to open Workspaces Activate the Analysis Instrument Settings l template tool Experiments Analysis templates and or Data files dependi
110. ect E Experiment Samples Sample ID Description Flow rate i Low mi Medium High fa Fickup and measure Liquid sensor E Mix sample Mode Standard A Uptake volume 150 H E Sample volume 200 wi E Annotations Autolabel Settings E Custom Express Type Setup Mode 3 Click the Type pull down list and select Setup Similarly choose Calibration from the Mode pull down list 4 Click on the Start Measurement button gt This will start the calibration process 4 1 3 Setting the dilution and mixing of the calibration beads prior to calibration Dilution of the calibration beads can be performed by the MACSQuant Instrument 1 Select the Autolabel tab within the Experiment tab 2 Click lt add gt This will introduce a Reagent dialog box 44 3 Select S1 and prebuffer and adjust the dilution appropriately The buffer should be set at 10 1 with no incubation time 4 Follow the steps described for manual calibration 4 2 Compensation of the instrument settings 4 2 1 Compensation of spectral overlap A proper compensation of the instrument is crucial for the optimal acquisition and display of data obtained by flow cytometry Compensation essentially accounts for the inherent overlap in emission spectra observed between different fluorochromes This fluorescence spectral overlap or spillover will result in the detection of individual fluorochromes in more than one
111. een and if required See below for more 108 4 Click Apply to implement changes Click OK close the window To assign a default naming conventions to user files and folders 1 2 3 4 Click Edit Options and Files Boc ll M m Users Experiment aii Public global aa Annotations Private user m Software Keyboard Logon name Timers Public backup Acquire J Eport Py Logon name Statistic FCS Private backup Backup 4 Regions Path Project Date P Windows File Initials Date Views Figure 7 16 Changing the default naming convention for user folders and user filenames Path The default naming convention for user folders is Date i e new folders are creating according to the system date YYYY MM DD File The default naming convention for user files is lnitials Date i e new files are creating using a prefix of the users initials followed by the current system date For example filename srv2008 12 10_2063 013 ori was created by user Srv on the December 10th 2008 The remaining part of the filename was defined by the user Data files ary2008 12 10_ 2063 01 3 ori sryv2008 12 10_ 2063 01 4 neg srv2U08 12 10_ 2063 015 pos Change the values between and as required Click Apply to implement changes Click OK close the window 109 To assign user permission to user groups 1 Click Edit Options and Access F Options
112. eling be performed or is sample analysis only required e Yes autolabeling is required Up to four MACS Reagents can be placed on the MACS Reagent Rack 4 for o Magnetic autolabeling of rare cell populations for subsequent pre enrichment and analysis 59 O Fluorochrome autolabeling of specific cell populations e No autolabeling is not required If samples are manually labeled no MACS Reagent Rack is required For manual labeling follow the labeling instructions in the corresponding data sheet 3 Are rare cells being analyzed e Yes rare cell analysis is required O Refer to chapter 6 for information on using the MACSQuant Instrument to enrich rare cells for analysis using MACS Technology Rare cells can be automatically magnetically labeled and enriched by the MACS Cell Enrichment Unit Depending on the selected analysis mode the enriched and non enriched fractions can be subsequently analyzed by flow cytometry A MACS Column must be installed for pre enrichment Refer to the user manual for instructions for using the MACS Enrichment Unit and MACSQuant column MACS Control Antibody Cocktails are available for automated and reliable rare cell analysis See http www miltenyibiotec com en NN_662_MACS_Control_Antibody_C ocktails aspx for a list of available products e No rare cell analysis is not required O No pre enrichment needed Having addressed the above questions the experiments can be easily de
113. ely use the keyboard shortcut Ctrl Alt R 3 Select sample positions for grouping These sample positions must be in a column for example BOO0O000 OO0000 i K CI O00000 Figure 5 15 Only samples in a column can be selected In this example column 1 was selected and adjacent sample positions Al B1 me 4 Click Group Group 5 Enter the sample information using the Experiment tab File Edit View Mode Analysis Window Help Ya NN la eee Samples Experiment Tools Channels Rack Chill 5 tube rack x File adm2009 09 10 001 v a Project CD14 Analysis admin Qm EE el e e O HSOSO Description Samples 1 amp 2 were grouped Liquid C Mix sampl Modi Standard v Uptake volume Ot LJ Sample volume 200 ul EJ oo o OO Figure 5 16 The above sample information applies to group 1 rack coordinates Al B1 only 80 6 Click on additional desired rack positions to perform further grouping Add sample information as required a aaa CN 6 z SA N AA H cg S Les as H Sf N Aa Fy 7 Close the Rack dialog box by clicking x 81 6 MACS Pre enrichment with MACSQuant Instrument The inclusion of the MACS Enrichment Unit within the system permits the magnetic enrichment of cells in situ prior to fluorescence analysis Th
114. ented as dot plots density plots histograms or statistical tables 1 2 2 MACS Control Applications The quality of cell separations using MACS Technology can be easily and rapidly assessed by the MACSQuant Instrument Customized measurement and analysis protocols can be created for the fluorescence analysis of cells while antibody labeling processing and sampling of cells can be performed in an automated fashion under the control of the MACSQuantify Software Specialized MACS Control antibody cocktails are also available for the multi parameter analysis of certain cell types such as CD14 monocytes and CD19 B cells 1 2 3 Rare cell detection The inclusion of the MACS Enrichment Unit within the system permits the magnetic enrichment of cells in situ prior to fluorescence analysis The MACS Enrichment Unit and the MACSQuant Column allow the possibility to reduce the number of cells analyzed to characterize the rare cell population of interest This is particularly useful for the analysis of cells present in low abundance such as stem cells dendritic cell subsets or natural killer cell subsets 1 2 4 Absolute cell quantitation The MACSQuant Instrument employs a robotic needle arm to acquire cell samples and to apply the sample into the flow cell The robotic arm provides the advantage of automation and the ability to sample a specific volume The MACSQuant Instrument can therefore count an absolute number of cells per uL volume of sample e
115. er manual 2 Adjust all PMT voltages for the cell sample e g FSCvSSC 3 Open a display template Plot3 by selecting the es icon 4 In the left dot plot set up to visualize FSC on the x axis and SSC on the y axis 5 Inthe right plot window change the plot type to histogram by selecting it from the drop down menu under the button 6 Set up to visualize the first fluorochrome requiring compensation e g FITC in the histogram 7 Inthe Experiment tab program all necessary paramenters such as Sample ID and uptake volume 8 Open the 8x8 7x7 compensation matrix by clicking the instrument settings icon and selecting the compensation tab 9 Place the first single stained tube e g FITC stained cells into the single tube holder 10 Start acquisition 11 When events start to appear on the plots pause the measurement by right clicking the stop button and choosing pause 12 Draw a scatter gate around the population of interest within the FSCvSSC dot plot This will be P1 13 Display only events in P1 in the histogram plot by selecting P1 from the drop down menu of the plot header 50 14 Draw an interval around the positive P2 and negative P3 populations Optional Rename P2 and P3 regions to positive and negative respectively by right clicking on the region and selecting region properties ao l 15 In the statistics table format properties under the i button Select to display P2
116. er mode Figure 7 30 Changing the default Views settings for plots histograms and tables 2 Select the required view category e Statistic click checkbox to display the header for the statistic table e Overlay color of histogram overlays can be choose e Histogram use drop down menus to select default values for displaying histograms Normalization Smoothing and Mode e Plot options 122 o Data All displays all acquired events by default Percentile displays a percentile value of the total acquired events by default namely 1 2 5 10 25 50 Fixed number displays a stipulated number of acquired events by default o Axes X Axis displays the selected axis scale by default Y Axis displays the selected axis scale by default Note It is recommended to use the default setting As required When performing data analysis users can modify the axis scale as required e Region functions select and highlight a function and using the and buttons move the region into the desired category Unused or In use Options Keyboard Timers Acquire Export Unused In use Backup gerah wae sa 7 CED Namie Regions Count sT Colors Countpl Up Windows Counter Pann Templates E Views Statistic Overlay Histogram Plot options Region functions Feature function x Region functions Sh o Name The name axis name o Count The actual total ac
117. ernal i No external media found To save an experiment definition 1 Highlight the Experiment tab on the Save window 2 Highlight the desired file location Private Public or External 3 By default the experiment definition will be saved to the user s private folder To save to an external drive highlight the External tab esas Note If no external media is attached to the MACSQuant Instrument or personal computer USB port the following error will be reported External D No external media found 8 7 Defining an experiment in Express mode A work through example In the following example three samples were placed in rack positions Al A2 and A3 of a Chill 5 Rack It is intended that sample positions Al and A2 will be analyzed using the MACS Control CD14 Monocyte Cocktail Sample position A3 will be analyzed using the MACS Control CD4 T Cell Cocktail 168 1 2 Note Ensure that the instrument is primed and calibrated Check that adequate reagents and buffer volumes are provided Ensure that the Definition tab is activated Definition Select Chill 5 tube rack from the Rack drop down menu u 5 5 mm fae a cee G Definition rack Flename samp _ Mode Figure 8 11 Selecting the Chill 5 Rack Left click once on rack coordinates Al and A2 Figure 8 12 Measure and select The settings for sample positions Al and A2 may be modified e g a la
118. etting and Analysis template 7 The samples have been assigned to a rack positions and defined for analysis 78 5 5 3 Rack configuration and sample grouping Sample grouping can be made before acquisition or afterwards during data analysis Refer to section 7 16 for information about sample grouping after data analysis What is the benefit of sample grouping The maximum sample volume that can be acquired in a single step by the MACSQuant Instrument is 450 uL There are occasions when the sample size is of course greater aliquots of the sample must therefore be spanned over two or more tubes By grouping these samples the acquired data will be consolidated into a single file on the hard drive which can also be analyzed in a single data file or analysis plot O 1 2 3 4 5 6 pp i 00000 e ee A samples 6000000 can be st er TF N analysed together c N 8 a rN E a FS Sa P See es AAAA Z OBOOOO0O eo Newt Net Ne uh Group Three groups created analysis folders created Figure 5 14 Schematic of the grouping process 79 To group samples 1 Using the Experiment tab select the desired rack from the drop down list Experiment Rack Chill 5 tube rack Single tube rack Fil 5 Chil 5 tube rack Project Chill 15 tube rack Chill 50 tube rack J E well Description Samples 1 amp 2 were grouped Sample ID 2 Click to open the corresponding rack dialog box Alternativ
119. f files during backup 4 Click Apply to implement changes Click OK to close the window 118 To assign default drag and drop properties for regions 1 Click Edit Options Software and Regions Options Users i Ee periment E Instrument Software Keyboard Drag amp drop Timers Create always link Acquire a E Export Change always ask w Backup B R egion Colors Views Regions Figure 7 27 Changing the default settings for drag and drop of regions 2 Use the drop down lists to activate deactivate a feature e Create when creating a new region e Change when changing a region 3 Click Apply to implement changes Click OK close the window To assign default color properties for regions 1 Click Edit Options Software Regions and Colors Options Users i Experiment E strument E Software 1 E black E magenta Keyboard Timers E dakgreen E green Acquire Region colors Backup Regions E eo oo 10 Export E darkred E darkblue E darkcyan Templates E Views Cancel Figure 7 28 Changing the default color settings for regions 2 Click on the color panel button adjacent to the region that should be changed e g tek 119 Note Regions are assigned numbered in ascending order i e the first region to be drawn is assigned the value 1 the second is assigned the value 2 etc 3 Select a new color Click OK 4 C
120. fined using the Experiment tab Define an experiment as follows Rack 1 Click the Experiment tab 2 Select the rack type using the Rack drop down list If the checkbox to the right of the field is activated the instrument will automatically detect the rack that is placed on the instrument 60 3 See section 5 4 for information on how to define multisample experiments using the Chill Racks Samples Experiment Tools Rack Rack Single tube rack File Single tube rack Chill 5 tube rack Experiment Chill 15 tube rack Sample ID Chill 50 tube rack C JE well Description yy L 4 Optional Change the filename The filename is automatically created by the MACSQuantify Software using the following default nomenclature lt User initials gt lt Date YYYY MM DD gt In this example user CU created a file on 11 May 2009 Rack Rack Single tube rack L File CU2009 05 11 001 vja Experiment 1 Enter alphanumeric text for the Sample ID and Description for example Experiment Sample ID CD34 sample one C Description CO34 rare cell analysis C Flow rate 1 Select a flow rate Low Medium or High or activate the checkbox to use the autorate function and select the desired event rate for collection With this function the instrument will adjust between the low medium and high flow rates to achieve the desired event rate Low Medium sa
121. fter the column has been removed from the magnet Process of cell enrichment is accomplished by the MACS Cell Enrichment Unit and the MACSQuant Column Target cells labeled with MACS MicroBeads are enriched using MACS Technology Computer controlled high precision syringe pumps with Teflon seal plunger that drive fluids through the MACSQuant Instrument fluidics system High resolution TFT color touchscreen located on top of the MACSQuant Instrument The touchscreen is used to operate and monitor the instrument via on screen menus Threaded plastic connector with a square nut used to connect the tubings to the bottle closures the column the pump or valves Permanent set of Teflon tubing through which fluid circulates in the MACSQuant Instrument fluidics system Different acrylic tube racks are available with the instrument and are designed for optimal positioning of sample tubes They contain a coolant allowing racks to be pre cooled in the refrigerator for subsequent cooling of the cells during analysis The automated arm carries a needle port designed for computer controlled uptake of sample This needle is calibrated and self cleaned It is designed to move in the y and z direction Container for waste fluid The closure is equipped with a fluid sensor The closure the fluid sensor cable and the tubing connector are color coded red 187
122. iated with a data file 1 Click and highlight the Instrument settings tab Highlight the desired instrument setting from a Public Private or External source 5 Public Privat Btemal Delete Workspaces Directory Instrument settings Z Instrument settings Experiments Analysis templates Setting human PBMC Open Cancel Danis be Open _ 2 Click Open The file settings will be loaded 3 Open the desired data file s The files will be loaded and can be view from the Sample tab 4 Click on the Sample tab menu 145 5 Right click on the data file and select Apply instrument settings Samples Bperiment Tools Channels Sample Statistic Cells live 0 0 0 T0z2009 01 23 am Open Ctri 0O TO22009 01 23 Add Rs Import FCS file Export sample Resample Recompensate Apply instrument settings Apply analysis template Remove Export sample list Recompensate post acquired data It is possible to recompensate data that was already acquired This is of benefit if data was acquired using incorrect instrument settings The results can be reanalyzed using a different compensation matrix To recompensate saved data 1 Open the desired data file s The files will be loaded and can be view from the Sample tab 2 Click on the Sample tab menu 3 Apply instrument settings analysis template as instructed above 4 Click to view the instrument setting
123. ideally suited to benchtop operation within the laboratory Also the instrument has several design features that permit the fully automated processing of cell samples from sample labeling and mixing through uptake and magnetic enrichment to fluorescence analysis The MACSQuant Instrument can be optionally fitted with the MACS MiniSampler The MiniSampler is a motorized sample rack holder that can hold tube racks of varying formats including 96 well microtiter plates The fully automated uptake and processing of multiple samples is possible under control of the MACSQuantify Software thus permitting the user a hands free high throughput operation Automated maintenance procedures are also a design feature of the MACSQuant Instrument This includes different system wash programs before each measurement automatic priming of the instrument and programs for shutting down the instrument for overnight or long term storage After sample uptake the instrument can analyze fluorescently labeled cells using up to nine or ten optical parameters seven or eight fluorescence and two scatter channels The software provided with the MACSQuant Instrument can perform standard flow cytometric data analyses and illustrations including histograms dot plots density plots gating and statistical views Data can also be collected in terms of time area height and width During acquisition the data are automatically stored in user designated folders for
124. ight click on the file and choose recompensate A new file will be created with an underscore _ before the file name _MM2010 03 02 005 Statistic Cells oO 0 100 0 10000 1000 10000 To view the newly created file press the A button and then double click on the recompensated file All gates and statistics will be applied to the new file 15 Values should continue to be adjusted until the Median fluorescence values for the positive and negative populations are equal for the spill over channel 16 To avoid creation of multiple files continue to apply the recompensation to w z Samples Experiment Tools Channels Sample Statistic Cells i Figure 4 8 Sample list with selected Apply instrument settings File Edit View Mode Analysis Window Help yy lt Sh live oD ci MM2010 09 02 00 open MM2011 0 09 02 00 la MM2010 09 02 00 wM2a10 09 02 00 Add Chri o feal Import FCS file Export sample Resample Recompensate Apply instrument settings 4pply analysis template Remove Export sample list 4 5 4 Usage of instrument bank settings the original file This will rewrite the second file with the _ before the file name and changes will be immediately visible on the analysis page To facilitate measurement of samples it is possible to save the PMT and Compensation settings in an Instrument setting
125. iments Each user s settings are determined by the administrator at the time of the creation of the user profile Express mode users are allowed to perform only minimal alterations to settings To modify Express mode settings and or gain access to more advanced options the Custom mode must be used Note Sole Express mode users do not have permission rights to perform Calibration or Compensation These options are restricted to Administrators in Custom mode and are located under the Mode and Setup drop down list ZN WARNING Read the instructions in the chapter Warnings and precautions in the user manual before operation of the instrument When processing infectious radioactive poisonous or any other hazardous liquids always abide by the necessary safety precautions 8 1 Quick guide to the Express mode main workspace The Express mode main menu for the user Express User EU is represented below 155 mq Express User A EE E m amp e EU Logout Definition Acquisition Analysis Rack Single tube rack v Filename EU2009 09 28 001 mad MC_CD14_h v Figure 8 1 Express mode main menu Category icon Description Definition Definition is used to setup an experiment i e enter sample details such as name description and required analysis mode Acquisition Acquisition mode displays live data that is being acquired Analysis Analysis mode is used to analyze acquired data for e
126. inally MACS MicroBeads offer an extremely flexible tool for the pre enrichment of many cell types from many species through the coupling of different antibodies Several hundred reagents for the isolation of human mouse rat and non human primate cells as well as reagents for indirect labeling of many other cell types are available Visit www miltenyibiotec com to learn more about MACS Technology and the MACS MicroBeads that are available After magnetic labeling the cells are passed through a MACS Column placed in the magnetic field of a MACS Separator Non labeled cells flow through and labeled cells are retained in the column and can be released after removal of the column from the magnetic field Thus labeled cell fractions can be efficiently isolated with MACS Technology The entire procedure is fast easy to handle and gentle on cells leading to the enrichment of cells that can be immediately analyzed The MACSQuant Instrument is equipped with the MACS Cell Enrichment Unit in order to provide a fast and easy way to analyze a rare cell population using MACS Technology The MACSQuant Column 130 094 458 when properly seated in the MACS Cell Enrichment Unit isolates up to 5x106 magnetically labeled cells If pre analysis enrichment is desired cells can be labeled with MACS MicroBeads separated using the MACSQuant Column and then directly analyzed by flow cytometry in a fully automated fashion Pre enrichment of target cells by MACS Te
127. is R4 MACSC b L D vja 1 100 vja 5 vis R5 3 MACSG a E L 0 vja 1 100 vio 5 vio R6 MACSQua ation Be i vio 1 100 vio 5 vio R7 alibrat ant Ca Be z i 0 vja 1 100 vla 5 vio S1 Buffer A 0 vie 10 1 viol via S52 e fer A 0 vja 10 1 vja s vja Apply Cancel Settings tab 1 Select either an Express or Custom mode of analysis Annotations Autolabel Settings Annotations Autolabel Settings Custom C Express Custom Express LJ Instrument setting Type Analysis Y C Analysis template Mode MC_CDi4_h lv C Gate C Events 10 000 IL For Custom mode analysis click the checkbox associated with the Instrument settings Analysis template Live gate and Events options to activate these features Express mode can be used for either calibration and compensation processes or with Miltenyi MC Cocktails for rapid and easy analysis of cells e Instrument setting previously saved instrument calibration and compensation settings can be loaded and applied by checking the adjacent box e Analysis template previously saved cell analysis templates can be loaded and applied by checking the adjacent box e Live gate check the adjacent box to activate live gating a live gating strategy can be saved as an analysis template for future use e Events check the adjacent box to stop data acquisition after a defined number of events is obtained in this example 10 000 events Note It is recommended to limit measurement by volum
128. is templates consist of a plot template new analysis window and if required a gating strategy Analysis templates can be created post acquisition or during acquisition in Live mode To create a useful analysis template whether in live mode or post acquisition the Experiment settings must have been correctly defined Refer to chapter 4 5 4 for more details An analysis window can be saved as an analysis template Click the New analysis window icon or use the file menu option Window and New analysis window to create a new analysis window 124 To create a new plot window 1 Click the icon or use the file menu option Window and New analysis window Figure 7 31 Available analysis templates are displayed by the Create New Plot Window 2 Click on the required analysis template 3 The analysis template will open as shown below Omaa o Note Multiple analysis windows can be opened These can be of single or multiple experiments Note If multiple analysis windows are open use the top menu bar icons to display the previous and next analysis window 125 7 14 2 Choosing a display format for plots histograms and Statistics T s Figure 7 33 Nine analysis templates are available
129. ktail human MC CO4 T Cell Cocktail humar MC CO34 Stem Cell Cocktail human e Time For autolabeling an incubation time is given The recommended incubation time is automatically shown in a black font type Experienced users may wish to change the incubation time using the adjacent arrows via note that non recommended times will appear in a red font type e g Ei e Titer For autolabeling a recommended label to sample titer is given The recommended titer is automatically shown in a black font type Experienced users may wish to change the titers using the adjacent arrows l note that non recommended or changed titers will appear in a red font type e g mega e Order Signifies the order at which this reagent will be used during cell processing 5 3 1 Selecting and assigning reagents manually using the MACSQuantify Reagents dialog box Note It is recommended to use the barcode reader to scan reagents refer to section 5 3 2 This protects the user against making incorrect reagent entries However if the reagent label or data sheet insert is damaged it may be necessary to manually select reagents using the Reagents dialog box 1 Place a reagent onto the reagent rack noting its position 2 Click Edit and Reagents 68 Note It is also possible to activate the reagent management window by checking an Autolabel lt add gt box located in the Experiment window Annotsior Fatidabel Settings fay MC CD14 C c
130. kup place 2 MTT autocomp mqd O eA Colin comp mqd ia F O comp APC MJ2009 07 03_2112 005 fcs CO eA comp PE MJ2009 07 03_2112 003 fcs C L comp PerCP MJ2009 07 03_2112 004 fcs Analysis templates Instrument settings xi Experiments Ey Other files gt 2 0 files selected 0 Byte Log files Delete Cancel 175 13 If using a network location to transfer file enter password when prompted 14 In the file folder window select the files or folders to be copied Data files as well as other files e g instrument settings and templates can be copied 15 Select copy 16 When all files are copied a report dialog box appears You can close the box to perform other copy or deletion functions 17 If using a USB thumb drive the option to close and eject is available Close and eject will allow safe removal of the USB device If eject is not chosen at this time go to Tools tab and select remove external media to stop the USB device safely 18 To remove files that have been copied the delete button in the copy dialog box can be used Folders or individual files can be deleted The fcs file format can also be deleted by this function 8 11 Exiting from the Express mode Logout 1 Click the Logout icon 2 If prompted to continue click OK The software will return to the login menu MACS Quantity Figure 8 20 Login menu 176 8 12 How to close the MA
131. l be increased by the same amount Thus the cell populations when using the same type of cells will be always in the same location in the plot even if the laser power already declined The bank settings are set up and controlled in the Calibration Settings window that can be found under Edit in the menu bar 56 Setup of bank settings 1 Have the instrument setting you want to have as a bank setting currently active 2 Open the window Calibration from the menu found under Edit 3 Select one bank 1 5 not the Default bank 4 Enter the desired bank name in the field calib name and click on Create 5 A pop up window confirms the generation of the bank settings files 6 Checking the active button enables the update of this bank setting with each PMT calibration 7 Clicking of the Load at Login button will set this bank as the default setting loaded at login into the software Example Current default calibration setting B Instrument settings MACSQuant Analyzer 10 34V via HMMM 430V via 368V via 432Vi via 422V via 538 V vja 378 V via 458 V via o E 386V via 526V via BB 6 70 via Specific instrument setting for assay E Instrument settings ACSI zer 10 2 x J FSC Trig li 30V via SSC Trig li B1 Trg B2 Tr B Tr B4 Tr 4144Vi via 368 V via 432V via 3 S 8S A A 422V via EM SC
132. l be used Events The events checkbox was not selected Absolute cell counting is therefore available since you will not acquire data after 10 000 events Rack configuration Samples in row A A1 to A4 will be measured Sa ae J a Autolabel tab MACS Control MC CD14 Monocyte Cocktail was selected and the vial placed on rack position one Table 5 1 Example of an experiment definition for analysis of four samples with the MACS Control CD14 Monocyte Cocktail 64 5 1 Switching to Express mode from Custom mode For more information on using the Express mode user interface see section 8 1 In Custom mode click Express mode button in the top right hand of the navigation bar im a lS home Figure 5 1 Switching to Express mode from Custom mode 2 The MACSQuantify Software window will change to the Express mode Note If windows are active in the Custom mode e g analysis window the user will be prompted to confirm this action Click Yes to continue and No to cancel the switch Note Any active work will NOT be transferred to the Express mode All data or settings must be saved before switching to Express mode 5 2 Printing in Custom mode The MACSQuantify Software uses installed windows printer drivers to print active workspaces Note The HP Universal Print driver has been installed on the MACSQuant Instrument and has been tested with the following printers Hp Laserjet
133. later analysis These folders are assigned for each user as either private or public access by the MACSQuant Instrument administrator The MACSQuant Instrument can also optionally perform pre enrichment of magnetically labeled cells before flow cytometric analysis This feature is based on the renowned MACS Technology and is particularly useful for the analysis of rare cells labeled with MACS MicroBeads Operation of the instrument is extremely simple through the use of the TFT color touch screen and intuitive screen menus built into the MACSQuant Software The user has the option of performing simple analyses pre programmed into the software using the Express mode or of customizing sample analysis protocols and automation programs using the Custom mode Data analysis using a variety of display options and functions can be performed on the instrument and the software has been configured for user friendliness and to provide highly flexible functionality Finally standard MACSQuant Buffers and Solutions which are directly attached to the instrument are sterile ready to use and designed for optimal instrument performance The instrument also provides a color coded LED warning system which illuminates the fluid containers to inform the user when buffers need to be exchanged or waste removed 1 2 Applications The MACSQuant Instruments are more than just flow cytometers The MACSQuant instruments feature a multitude of design features that enhanc
134. lear All sample menu Clear Selected B Cy button N Note In order to set all rack te positions to allow Measurement and modification of the experiment strategy e g labeling Click Select All Single right click Selection deselection of an entire on column header a ene eS sample column a ee incol N Single right click 1 a2 Selection deselection of an entire 3 4 a 6 on row sample row In this example ser A fh ON Row A is selected for sample Clear Row PA N K A y a g ee _ s _ labeling and measuring f Deselect Row Row B is selected for sample measurement only Single right click o 1 2 3 a 5 68 Right click over a single rack over a single rack 0O 0O QO position to completely clear this N O position In this example position position ae ss F sai A2 will be cleared Table 8 4 An overview of the possible configurations for rack positions 161 8 5 2 Sample ID and Description The Sample ID and Description boxes are text fields in which alphanumeric characters may be entered in order to name the sample Sample ID and provide a more extensive description Description about the sample Both fields are optional jeja m amp w O Help Custom Definition Rack Chill 5 tube rack x es C amp C O O00000 Sl O00000 BAE LA EA EI ke C d Figure 8 5 Location of the Sample ID and Description fields
135. lick Apply to implement changes Click OK close the window To assign default properties for Windows and Window templates 1 Click Edit Options Software and Windows Options Users gt Experiment El Instrument El Software Delete Always ask Keyboard Timers Acquire Export Backup Regions Colors Se indos Templates E Views Windows Figure 7 29 Changing the default color settings for windows 2 It is possible to configure a warning prompt when users click in order to close an analysis window e Never ask the window will immediately close when is clicked e Always ask the user will be prompted to Close the current window when is clicked 120 3 Click templates to configure the layout of window templates Options E Users i Experiment E Instrument ial Software Rows a wi al Cols 3 vljal Keyboard i i i l Timers Acquire a Export x Backup E Regions Colors Windows emplates Templates 3 PPS abec 4 PPPP abed 4E PPPS abedddddd 6 PSPSPS abbeddeff 6a PPPPPP aabsacdet 9 PPPPPPPPP abedefghi Plots types are assigned the following nomenclature P Dot plot D Density plot H Histogram T Text S Statistic N None blank The total number of plots in a plot layout is given by a number from 1 to 16 The position of a plot in the plot layout is assigned by letters abcdefg etc where positio
136. ll plots _Sample grouped into B grouping a single file Figure 7 52 Schematic of the grouping process 151 To group samples 1 Click File Open and navigate to the files that must be grouped Highlight the files and click Open am Publica Privates Etemal Delete Workspaces Directory Data files 6 Data files TOz2003 01 23_53 003 mad gj 2011 07 15 T02z2009 01 23_53 010 mad T0z2009 01 23_53 011 mad L Files 0z2003 01 23_53 003 mqd TOz2009 01 23_53 010 mqd TOz2009 01 23_53 011 maqd Ee Cancel Figure 7 53 Three files were opened for grouping 2 Using the Samples menu Eme h highlight each file that must be grouped 3 Right click and select Group z2009 01 23 T022009 01 23 53 0 T022009 01 23 53 010 100 0 30000 moe T02z2009 01 23 53 003 100 0 30000 Export sample list Figure 7 54 Left Selecting three data files for grouping Right The resulting grouped file is highlighted 7 17 Export sample list It is possible to export the sample list to an excel table The sample list table is a summary of all samples with corresponding statistics To export a sample list 1 Click on the Sample menu 2 Highlight the sample files that must be exported Right click and select Export sample list 152 File Edit View Mode Analysis Window Help admin ESS le SES ES EA NO NEE EE EO
137. mport FES file T Export Sample Resample Recompensate Apply instrument settings 4poply analysis template Remove Export sample list Figure 7 5 Applying analysis template saved within mad files 94 6 If files were acquired using MACSQuantify Software Express templates there will be an additional option to view with Express Analysis xxx e g Express Analysis MC_CD1 9_h 7 Choosing this will open up the Express template with automated gating and analysis This template can also be modified by deselecting the Analysis Mode icon 7 8 Opening files 1 Click to open the Open window External Delete Directory Workspaces A pii By Instrument settings by Experiments Reagents by Analysis templates ia Data files Workspace Figure 7 6 Custom mode users and administrators are able to open Workspaces Instrument settings Experiments Reagents Analysis templates and Data files 2 Click on the desired tab for example the Experiment tab or Data file tab to open an experiment definition or data files respectively 3 0 9 8 ay 3 a 2 2 g 1 8 a5 m 2 G S amp D 7 D a g Py T al a 5 g Eg a gt f g fa io 3 amp iz mj a r lt o E ry ka Be d a g T DU Experiment CD34 Stem Cell Analysis Data files _stv2009 02 04_2062 054 pos
138. n StdDev standard deviation CV coefficient of variation Min minimum Max maximum Median and Modal values Table 7 3 An overview of the Properties settings for the statistic option Overview of the Properties settings for text A variety of display settings for the text option can be modified using the Properties tt i 7 option from the drop down menu 1 Click the icon located adjacent to the text box 2 Click Properties M The properties window will appear Tab Category Property Associated screenshot Description option ee Free text or a MACSQuantify Software text Sot Tet script can be entered into the Script Text field Scripts are based on HTML hypertext mark up language and can be Text Script Free written to automatically display statistics about each region or gate Specific regions can be removed from or added to text table by using the overlay tab A script was used to create the following text table OK Cance Apy BB Properties Overlay Overlay Population Overlay Population 1 Population 2 Population 3 Population 4 Text Overlay m m a sr 2008 12 10_2063 015 pos P3 P4 m s Apply smer ATAA U A0 T pna Legend Neginn Papulnlinn aunt i Dehe exe uann TI PitP2 Miale erk ug ley 1178 PIFAP Grandozyte sxdusio Tor PISPAPS P CO Moncey se Treo Scripts are provided by MACSQuant Instrument speci
139. n A is at the top left hand corner of the plot window and the last letter used is at the bottom right hand corner of the plot window Letters can also be reused e g aabb this allows the user to assign a plot type over two or more positions For example Plot4 can be described as 4 PPPP abcd That is 4 plots will be assigned to the total workspace Each plot will be dot plots P abcd denotes that each dotplot will be placed in each corner of the workspace Another example Plot3 can be described as 3 PPS abcc 121 That is 3 plots will be assigned in total Of which two will be dot plots PP and one will be a statistical table S The dot plots will occupy the upper left and upper right corners of the workspace ab The statistics table will occupy the entire lower half of the workspace cc Note Hyphens are used to separate the conditions used to describe plot layouts e g 3 PPS abcc Only hyphens may be used 4 Configure the dot plots as necessary Click Apply to implement changes Click OK to close the window To assign default properties for displayed views of plots histograms and tables 1 Click Edit Options Software and Views Options Instrument Software Keyboard Timers Acquire Text H black 4 Export Backup Background background Colors Statistic Overlay Histogram Plot options Region functions Feature function C Multilay
140. n bandpass filter PE emission l spectra ii filter f spectra gt J gt a gt rn Bleed of FITC fluorescence into PE PMT A N 450 500 550 600 650 Wavelength nm Normalized excitation emission D D Ww Ww Bleed of PE fluorescence into FITC PMT gt N gt Figure 4 4 Emission spectra of FITC green solid line and PE red solid line Green hashed area of the FITC spectra represents the amount of FITC signal detected by B2 Red hashed area of the PE spectra represents the amount of PE signal detected by B1 Spill over is determined by acquiring single fluorochrome stained samples and viewing the fluorescence signal versus all adjacent detection channels With properly compensated instrument settings the median fluorescence intensity of the spill over channel of the positive and negative population is equal Figure 4 5 B When the instrument settings are undercompensated the median fluorescence intensity of the spill over channel of the positive population is greater than that of the negative population Figure 4 5 A When the instrument settings are overcompensated the median fluorescence intensity of the spill over channel of the positive population is less than that of the negative population Figure 4 5 C Using statistics to adjust compensation to result in equivalent median fluorescence intensities is preferred A Undercompensated B Compensated C Overcompensated
141. nal intensities can extend over several orders of magnitude As a consequence a logarithmic scale must be used to allow visualization of all populations The impact of selecting an appropriate scale is exemplified by Figure 1 5 below In this example a sample was analyzed which contained a population of white blood cells labeled with the fluorochrome phycoerythrin PE A linear scale was used to display a dot plot of forward scatter vs side scatter A and to display a histogram of PE fluorescence intensity versus cell count B In B the signal intensities of non fluorescence cells and fluorescence labeled cells are squeezed together To separate the signals a log5 scale was used Figure 1 5 C revealing two peaks the left peak is attributable to background fluorescence whereas the right peak is due to cells labeled with PE It is important to note that the same data can look quite different depending on the scale used however the values of the data points have not been altered Further as the MACSQuant Instrument acquires data in a digital format some fluorescence intensities may be assigned a value less than zero Data values less than zero may not be displayed properly using a conventional logarithmic scale although all calculated statistics will be correct This is a general feature of more advanced digital flow cytometers To overcome this a hlog or biexponential scale may be used D In an hlog scale the upper values of the scal
142. ng on user access rights set by the administrator Click to save Workspaces H Scroll through samples Instrument settings and listed in the samples Experiments depending on user window access rights set by the administrator Backup or transport data Delete a region that was created Activate the reagent in a dot plot or histogram barcode scanner O Draw a region in a dot plot i e Open the rack dialog box 3 to define an area of interest ji i 4 Open the instrument EMIpSe TALADI Aa settings dialog box polygonal regions can be drawn Ci Activate touch screen keyboard Draw a quadrant in a dot plot Open help file and manual Draw an interval in a histogram Switch to Express mode Open a new analysis window Logout user from session Close analysis window Main instrument control Click to switch between Acquisition mode or Data analysis mode The instrument may also be switched off using this button Scroll through open analysis admin Name of user in the top windows in a reverse and right corner in this forward direction example the administrator admin is logged in Table 3 1 Quick guide to the MACSQuantify Software top menu bar icons 28 Quick guide to the MACSQuantify Software menus File GE Edit View Mode Analysis Window Help New workspace Ctri N Open Ctrl 0 Save Ctri Import FCS file e n Copy Logout Logout Quit Quit W Choose
143. o select a rack configuration perform the following steps 1 Click on Definition to define the experimental setup 2 Choose the rack format using the Rack drop down list In this example Chill 5 tube rack was chosen for the measurement If ojaja m E ro en n 0 Definition Figure 8 4 Selecting the Chill 5 Rack for multisample labeling and cell analysis 3 For details on how to choose the correct rack format refer to Table 8 2 Rack type Slots Option on Corresponding MACSQuantify Rack MACSQuantify Rack drop down list graphic Single 1 x5mL Single tube rask w Not applicable tube rack Chill 5 24x5mL Eee y Chill15 15x15 mL OO oie 5x5 mb rattan tae So Sa bee yy JOU 159 Chill 50 Chill 96 96 well microtiter plate Chill 50 tube rack v 96 well v Table 8 2 Overview of the various rack types that may be used with the MACSQuant Instrument An appropriate rack should be used depending on the sample number and volume Configuring the sample rack 1 Click on a sample position using the left mouse button This will allow you to select deselect and activate deactivate the sample Refer to Table 8 3 fora summary of the potential rack configurations for single sample positions For some express modes grouped samples are required After selecting the desired sample position click the group button to designate this User action with left mouse
144. oard Shutdown timer 5 min C Acquire Needle priming time 20 sec C i eer Shutdown default behavior ackup Regions Analysis mode Instrument off Windows Views OK Cancel Figure 7 22 Changing the default timer settings 2 Use the slider bar or text field to change the timers for the Standby timer instrument goes into standby after the stipulated time of inactivity Shutdown timer instrument performs a controlled shutdown after the stipulated time of inactivity Needle priming timer instrument performs an automated needle priming at stipulated intervals 3 Shutdown can be defined as Instrument off default or Analyse mode the instrument switches to Analyse mode and does not power off 4 Click Apply to implement changes Click OK close the window 5 Click Edit Options Software and Timers To assign default settings for acquire Note Feature only applicable to the MACSQuant Instrument This function is not available to MACSQuantify Software installations on personal computers 114 1 Click Edit Options Software and Acquire i Eeperimert E Instrument Remote Control E Network setup E Software H Keyboard E Regions Windows fl Views Acquire Reset sample on clear view Live events 5 000 al lt gt C Export as FCS Show volume progress in pl Apply express analysis Figure 7 23 Changing the default acquire settings 2 Use the check boxes to activate a feat
145. of multiple cellular antigens These filters create fluorescence channels which is monitored by a photomultiplier tube PMT Each PMT which is located after a filter set will amplify the signal of the detected light Therefore the laser will excite a fluorescence marker on the cell which will be deflected by the cell and collected at a 90 angle This deflected light will pass through the appropriate filter and the resultant signal will be amplified and reported by the flow cytometer software i e the MACSQuantify Software It is strongly recommended to assign meaningful names in the software for each photomultipler tube detector before beginning an analysis i e the naming nomenclature must correspond to the fluorochromes used for cell staining For example FITC fluorescein isothiocyanate has an emission maximum of 521 nm in water and is thus detected in the green fluorescence channel B1 525 nm with a bandwidth of 50 nm The MACSQuant Instruments are equipped with three lasers for measurement of up to seven or eight fluorescence channels and two scatter channels FSC SSC The MACSQuant Analyzer 10 is equipped with an additional fluorescence channel for detection of signals such as VioGreen or Pacific Orange The MACSQuant VYB is a three laser instrument with optimized lasers and filters for detection of fluorescent protein expressing cells Fora list of representative fluorochromes and their respective detection channels on each type
146. omatically saved as the default settings 5 The calibration results for each channel are presented as dot plots histograms and as a tabulated summary on a two page two screen report Successful 42 calibration for each channel is indicated by a green checkmark To view all calibration dot plots and histograms click Next screen or Previous screen File Edit View Mode Analysis Window Help agal lt e e Tee wlio Owaa if ai ai ac ml Ni quiet He ear Time Volume Calib ok 1 day old 0 File Edit View Mode Analysis Window Help LO epee Single tube rack adm2003 09 01_ 2022 003 SJA 4 Figure 4 3 Successful calibration of the MACSQuant Instrument as shown by an array of histograms upper and associated summary table lower 43 4 1 2 Performing manual calibration Custom mode users and administrators can perform manual calibration as follows Note The calibration beads must be pre diluted and mixed before performing this procedure The MACSQuant Instrument can perform pre dilution and mixing of calibration beads see section 4 1 3 for more details 1 In the Custom mode select the Experiment tab on the left side of the screen 2 Select the Settings tab in the lower section of the panel and click on the Express radio button Tools Channels Experiment Rack Single tube rack El File adm201 1 06 30 on v4 E Proj
147. ommended and can be performed automatically on the MACSQuant Instrument 4 1 Calibration of the instrument settings In flow cytometry fluorescence intensity is used to distinguish between positive and negative populations of particles The reproducibility and stability of the fluorescence signal over time is of vital importance In order to ensure a stable measurement that is independent of time and the specific analyzer instrument calibration is performed Fluorescence calibration curves are calculated by using standardized fluorescence MicroBeads that have predefined sizes and fluorescence intensities A linear regression equation is calculated from the instruments response in mean or modal histogram channel values to these predefined values 4 1 1 Performing a fully automated calibration 1 Prior to beginning calibration ensure that the single tube holder is correctly attached Figure 4 1 The single tube holder and orange acquisition button 2 Activate the reader by clicking on the Barcode icon lll and present a vial of MACSQuant Calibration Beads in front of the barcode reader To proceed with the calibration process select Yes 41 3 Follow the dialog box instructions i e place an empty tube into the single tube holder and dispense one drop of MACSQuant Calibration Beads into it Note Ensure that you mix the calibration beads prior to dispensing 4 Click OK to commence the calibration process The upt
148. ontinue Software registration i l jx Use following information or http wii cilterwibiotec con to register your copy of MACS Ouantihy Software version 2 1 1003 R Activation key 57339048 Enter your name and registration key User name Registration Figure 2 4 The software version and activation key are required in order to register the MACSQuantify Software 25 2 4 Login on to MACSQuantify Software Note MACSQuantify Software will operate for a trial period of 30 days without registration Refer to section 2 3 for information on registration of software 1 Launch MACSQuantify Software 2 Select Admin from the drop down list and click Login 3 Complete the password field Confirm your password in the field underneath Click OK 26 3 Custom mode The Custom mode is designed for advanced flow cytometry users Administrators and advanced users can use the Custom mode interface to create customized experiments ranging from sample autolabeling and uptake through data acquisition gating and data analysis to the generation of print ready results Custom users and administrators have advanced access to MACSQuant Instrument and software settings Administrators have additional permissions concerning setting user permissions and the management of Express and Custom mode users Both administrator and Custom user features are discussed throughout this chapter CZN WARNING Read the instructions in th
149. opyright 2011 Miltenyi Biotec All rights reserved Content Read the MACSQuant Instrument user manual before using the instrument 2 1 Introduction 8 telO O T gt ee ee ee re ee eee 8 1 2 APPLICATIONS 32S 23 S552 ee Se SS Se eee eee eee 9 1 2 1 Fluorescence cell analysis 9 1 2 2 MACS Control Applications 10 1 2 3 Rare cell detection 10 1 2 4 Absolute cell quantitation 10 1 2 5 Automated cell labeling and analysis 10 1 2 6 Flow cytometry an introduction 1 1 2 7 Displaying flow cytometric data 13 1 2 8 Analyzing flow cytometric data using regions or gating 17 1 3 MACS CELL SEPARATION 19 1 4 DESCRIPTION OF THE MACSQUANT ANALYZER MACSQUANT ANALYZER 10 AND MACSQUANT VYB MMMMMMMMMtMtMtMtMtMMM 20 2 Installation of the MACSQuantify Software 22 2 1 INSTALLING SOFTWARE ONTO A PERSONAL COMPUTER 22 2 2 REGISTERING THE MACSQUANTIFY SOFTWARE ON A PERSONAL COMPUTER 23 2 3 MACSQUANTIFY SOFTWARE REGIST
150. ot recommended to activate the Event limit setting H Event limit Limiting data acquisition to the number of events prohibits volumetric cell counting 3 Click Apply to implement changes Click OK close the window 7 13 3 Changing the default instrument options The default instrument options are the instrument name instrument features and instrument annotations Each is briefly discussed below To assign a default instrument name 111 1 Click Edit Options and Instrument E orton i Files a Users Access Experiment Name Annotations Re Hardware profile MACSQuant Analyzer x Keyboard r Timers Acquire Export Statistic FCS Backup 4 Regions Windows Instrument Figure 7 19 Changing the default instrument name 2 Enter alphanumeric text into the Name field 3 Click Apply to implement changes Click OK close the window To assign a default instrument annotations Instrument annotations are used to provide additional description of the eight analysis channels that are available on the MACSQuant Instrument Data analysis and interpretation is arguably easier when suitable annotations are used for example B1 channel could be annotated as FITC as this fluorochrome is detected in this channel 1 Click Edit Options Instrument and Annotations FSC FSC B3 PI PECy5 5 Software SSC SSC B4 PECy7 V1 VioBlue R1 APC B1 FITC R2 APCCy7 B2 PE OK Cancel
151. ples as well as analysis of the collected data Automation can further be extended to multisample processing when combined with the MACS MiniSampler for convenient hands free operation Needle arm Cell Enrichment Unit MACS MiniSampler Figure 1 8 Front image of the MACSQuant Instrument the access cover was made transparent for the purpose of illustration The MACSQuant Instrument Compact benchtop design Straightforward multiparameter cell analysis from simple cell counting to sophisticated flow analysis Absolute cell counting volumetric Highly sensitive detection of rare cells Fully automated multisample labeling and analysis 21 2 Installation of the MACSQuantify Software The MACSQuantify Software is installed on every MACSQuant Instrument and can be used both for sample acquisition as well as for analysis of data collected on the MACSQuant Instruments of from other flow cytometers To install the software for data analysis from a PC please follow this guide 2 1 Installing software onto a personal computer Note The recommended PC specifications to run the MACSQuantify Software are Operating system Microsoft Windows XP SP2 is a minimum requirement although SP3 is preferred Memory 1GB minimum Note MACSQuantify Software is only compatible with Microsoft Windows operating system Note Users must have administrator rights on their C drives to install the MACSQuantify Software 1 Inser
152. quired events or count o Count uL Number of acquired events per microliter o Count mL Number of acquired events per milliliter e Feature functions select and highlight a feature function and using the and buttons move the feature into the desired category Unused 123 or In use Options Keyboard Timers Acquire Export Unused Backup Mean Gl Regions StdDev Colors cy J windows bin Templates Max el Views Median statistic Modal Overlay Histogram Plot options Region functions Feature functiondkal Feature functions 3 After making the necessary modifications click Apply to implement changes 4 Click OK close the window 7 14 Data analysis in Custom mode Acquired data are displayed and analyzed by an analysis window Depending on which New plot window template is applied by the user analysis windows may contain dot plots density plots histograms statistics and text tables Several analysis windows can be opened at one time These can be of several experiments or of a single experiment with a complex gating strategy Gating strategies can be created during sample acquisition live and saved for future use or can be created post acquisition For background information concerning gating or defining regions of interest refer to section 1 2 8 7 14 1 Creating a new analysis template or analysis window Analys
153. r action to select multiple sample positions Single right click of the multiple sample menu button Single right click on column header Single right click on row Single right click over a single rack position Grouping function o i Select All Select Used Deselect All Clear All Clear Selected B I j Select Used in Col Deselect Col Clear Col Clear Selected in Col 1 2 3 4 5 6 Select Row Select Used in Row Deselect Row Clear Row NY Clear Selected in Row i ae d p D Fi A Clear Group D DACUK Use this button to change the settings for all rack positions Note In order to set all rack positions to allow Measurement and modification of the experiment strategy e g labeling Click Select All followed by Deselect All Selection deselection of an entire sample column Selection deselection of an entire sample row In this example Row A is selected for sample labeling and measuring Row B is selected for sample measurement only Right click over a single rack position to completely clear this position In this example position A2 will be cleared Only rack positions that are adjacent and in columns can be grouped To group several adjacent rack positions 1 Select the rack positions 2 Click Group Table 5 4 An overview of the possible configurations for rack positions To configure a sample rack 1 Click on
154. roBeads are not known to alter the structure function or activity of cells Enrichment with MACS MicroBeads is only achievable with MACS Columns such as the MACSQuant Column This column is specially designed with stainless steel spheres to allow a enrichment over several months of use once integrated into the MACSQuant Fluidics Use of MACS Technology for pre enrichment with the MACSQuant Column In a first step surface antigens are magnetically labeled in a highly specific manner with monoclonal antibodies coupled to MACS MicroBeads This is followed by 82 fluorescent labeling with necessary immunophenotyping antibodies After magnetic and fluorescent labeling the cells are loaded onto the MACS Column placed in the magnetic field of a MACS Enrichment Unit Non labeled cells are gently washed away from the labeled cells that are retained within the column s magnetic field The enriched fraction is then eluted from the column when the magnetic field is disengaged and flow directly to the flow cell for measurement As the MACSQuant Column and MACS Enrichment Unit is seamlessly integrated into the MACSQuant Fluidics the entire process can be easily performed with one programming step Pre enrichment with the MACSQuant Instrument has been successfully used to analyze endothelial progenitor cells in blood antigen specific T cells labeled with tetramers and circulating tumor cells in blood With a highly diverse portfolio of MACS MicroBeads f
155. rom which to choose there are many possibilities to increase the sensitivity of rare cell analysis Properties of the MACSQuant Column Capacity retains up to 5x106 magnetically labeled target cells Life span 3 months Sample volume in Experiment Tab Volume of sample to enrich on MACSQuant Column maximum of 5 mL Uptake volume in Experiment Tab Volume of eluted fraction to measure on MACSQuant Instrument maximum 450 uL Pre enrichment programs of the MACSQuant Instrument Enrich EnrichS EnrichS2 Designated sample volume is loaded onto MACSQuant Column Process description Negative fraction is washed away to waste Positive fraction is eluted and directed to flow cell Min max sample 25 uL 5 mL 25 uL 5 mL 25 uL 5 mL volume Cell separation 0 75 mL min 0 5 mL min 0 25 mL min rate Note Cells have to be labeled magnetically and fluorescently since they will be immediately measured after enrichment and can not be labeled after that 6 1 Utilizing the MACSQuant Column for pre enrichment a walk through example In this example the EPC Enrichment and Enumeration Kit human 130 093 477 in combination with the MACSQuant Instrument is designed for the enumeration of circulating EPCs cEPCs from peripheral blood cord blood or leukapheresis products as well as EPCs from bone marrow following the pre enrichment of EPCs For further information on this kit please visit http www miltenyibiotec com en PG_678_8
156. rror margin 5 This also means that multiple cell populations can be simultaneously enumerated within a sample after fluorescence staining and analysis For example the ability of the MACSQuant Instrument to provide absolute quantitation of cell populations permits the optimized enumeration of specific cell types using pre defined software analysis protocols and specialized antibody kits For example using the CD4 RTE Enumeration Kit 130 092 055 the enumeration of CD4 recent thymic emigrant RTE cells is possible from whole blood samples or peripheral blood mononuclear cells PBMCs Furthermore the automated processing of cells and the use of the MACS Mini Sampler facilitate the seamless incorporation of the MACSQuant Instrument into routine high throughput laboratory cell enumeration procedures Labeling reagents and software analysis can be customized to suit individual applications 1 2 5 Automated cell labeling and analysis Automation of cell sampling and analysis can be extended to include the labeling and dilution of cells with the MACSQuant Instrument With the use of the MACS MiniSampler in combination with the Reagent Rack and the Chill Racks the automated processing of up to 96 samples is possible 1 2 6 Flow cytometry an introduction Any given cell population can be defined by its individual expression profile of both intracellular and extracellular antigens These antigens can therefore be targeted for their detection an
157. rs can be entered directly into the field or by using the arrows 131 Axes Options Region functions Overlay X axis Regions Normalization Smoothing Mode Regions Functions Population Axes X axis x Options Regions All v A Normalization All This Smoothing None Options Regions All B Normalization None v Smoothing Area Options Regions All 1 Normalization ioe eE Smoothing Off a Mode or Light Medium Strong Options Regions All a Normalization None a Smoothing Ort 3 Mode Line 3 E Properties View Region functions Overlay Overlay Population liv i m Population 2 live m i C Population 3 live m Oo Population 4 live a Crea w The x axis scale of a histogram can be configured as follows As required automatically configured lin linear scale log2 5 logarithmic scales hlog biexponential scale No regions None all regions All or only the selected region This can be shown on the histogram The histogram graphically summarizes the distribution of a univariate data set Data can be normalized by area integral of total area under the curve or by height Algorithms can be used to smooth the histogram Light medium or strong smoothing is available Histograms can be display as a line chart or bar chart The latter is used to a greater degree
158. s of the company within the Miltenyi Biotec group which supplied the Product The Terms may vary by country and region Copies of these Terms are available on request or at www miltenyibiotec com Nothing in this document should be construed as constituting an additional warranty Miltenyi Biotec s product warranty only covers Product issues caused by defects in material or workmanship encountered during ordinary use as described in the user manual or other documentation provided by Miltenyi Biotec it does not cover Product issues not arising out of defects in material or workmanship including but not limited to Product issues resulting from failure to follow installation operating and or maintenance instructions or environmental conditions prescribed in this user manual or other Product documentation misuse abuse neglect mishandling unauthorized or improperly performed maintenance or repairs accident acts of God limitations of technology electrical current fluctuations modification of or to any part of the Product use of accessories spare parts and or consumables other than those recommended by Miltenyi Biotec or normal wear and tear Miltenyi Biotec s product warranty does not cover products sold AS IS or WITH ALL FAULTS or which had its serial number defaced altered or removed or any consumables or parts identified as being supplied by a third party those third party accessories or parts may be covered by a separate
159. s Experienced users can modify the Channel and Compensation values if required Click Apply to implement 146 changes to the compensation matrix Instrument settings FL Channel Matrix VioBlue 7 000 a ooa J ooo Ea 5 5 S 5 PIPE Ly5 5 ce 6 000 CJ 1 000 LJ 0 000 GJ PE Cy7 ra a 0 000 it Fo 1 000 it APC 0 000 L 0 000 i 0 000 L 0 000 LA APC Cy cs Fo c 0 000 LA 0 000 LJ R1 0 000 R2 0 000 0 000 I 0 000 I ono a cos a E it ut uw ci L 0000 J L 1 000 ooon 5 Right click on the data file and select Recompensate A new file will be created with the adjusted compensation values applied These files are prefixed with an underscore i e _srv2008 12 10_2063 018 pos gt Open Add Ctri O L3 IE Import FCS file Export sample D al Resample Recompensate Apply instrument settings Apply analysis template Remove Export sample list Figure 7 50 Recompensating a data file It is also possible to recompensate all data files that have been opened and are shown in the Samples window 147 7 15 5 Live gate What is a live gate Only events within a live gate are acquired and saved by the MACSQuantify Software Live gates are useful when large datasets are being acquired Since only da
160. s are to be backed up e always all files all files are always backed up there is no user prompt to verify this procedure e always data files only data files are backed up there is no user prompt to verify this procedure Files deletion e automatic automatically overwrite or delete files during backup e ask the user is prompted to verify deletion or overwriting of files during backup 106 3 Click Apply to implement changes Click OK to close the window 7 12 5 Configuring network settings administrators only In order to have remote assistance and data backup to a network location it is necessary to configure the MACSQuant Instrument network configuration The settings will be initially done by your MACSQuant Specialist It may be necessary to contact your local network administrator If there are any problems please contact Miltenyi Biotec Technical Support 7 13 Configuring the default user instrument and software options The Options menu is used to customize user software and instrument settings Custom users and administrators can customize software and instrument settings An explanation of the options menu follows 7 13 1 Changing the default user options Under Edit gt Options you are able to pre define the default setup for following processes User groups Express Custom and Administrator are managed using the Users menu File access permissions and the default location of files can be
161. s file This can be reloaded the next time the same or a very similar experiment is performed However since lasers decrease in intensity over time the long term usage of a defined instrument setting is critical The decline of laser intensity results in weaker scatter and fluorescence signals The original 55 strength of the measured signal can only be restored by adjustment of PMT sensitivity which actually means that new instrument settings have to be created The MACSQuant Instrument offers an alternative way in saving instrument settings which accounts for changes in the laser intensity Offset Stores the difference in voltage between default Creates the bank calibration setting and bank When activated settings Setting the bank setting will be loaded as default setting upon login Field for entering name of bank setting Bank setting will be updated with v SSC format lv vi Off every PMT W v2 Off calibration when W B1 OFF this option is F B2 Of Sera checked FI B3 i scale V B4 Off Off Off Determines channel for primary Figure 4 9 The calibration settings window Bank settings The principle of these so called bank settings is that the voltage applied on each PMT depends on the current PMT calibration setting If the instrument increases the PMT voltage to achieve the optimal sensitivity during calibration the voltage setting in the connected bank setting wil
162. s from three human PBMC samples were enriched using the auttoMACS Pro Separator in combination with the Monocyte Isolation Kit Il It was important to evaluate the outcome of these cell separations using flow cytometry This can be easily and quickly achieved using the MACSQuant Instrument in combination with the MACS Control MC CD14 Monocyte Cocktail human Three samples were placed on rack positions Al A2 and A3 of a Chill 5 Rack Cells in sample tube positions Al and A2 were analyzed using the MACS Control CD14 Monocyte Cocktail in the Express mode Cells in sample tube position A3 were analyzed in the Custom mode using an analysis template The default instrument setting was used i e the most recent instrument setting which is defined as Default Banks See sections 3 and 7 for more information on instrument settings and analysis templates respectively 5 5 2 Rack configuration and sample definition 1 Choose Chill 5 tube rack from the Experiment tab Fie Edit view Mode Analysis Window Help f i TFA ca o len tT dga 2 al te a fe a E O Samples Experiment Tools Channels Experiment Rack Chill 5 tube rack v File adm2009 09 08 001 vja amp Annotations Autolabel Settings FLI VioBlue FL5 PE Cy7 FL2 FITC FLG APC FL3 PE FL APC Cy FLA PIPED O
163. t the MACSQuantify Software USB memory stick into an available USB port on the PC 2 Copy the folder entitled cap from the USB drive to the Windows desktop 3 Open the cap folder 4 Run file installCAP 5 Answer the command prompts Yes Y NON or Abort A as instructed below 6 At the prompt Do you want to install a new cap package Y es A bort Select Y to continue with the installation or A to abort the installation 7 At the prompt Install on MACSQuant Y es N o A bort Select N when installing the software onto a PC Select A to abort the installation 22 8 At the prompt Do you want to keep old configurations and settings Y es N o A bort Select Y when current software configurations and settings should NOT be deleted by the new installation Select N when current software configurations and settings should be deleted by the new installation Select A to abort the installation 9 At the prompt Do you want to keep all data files Y es N o A bort Select Y when saved data files should NOT be deleted by the new installation Select N when saved data files should be deleted by the new installation Select A to abort the installation 10 The program will automatically install the software using the previously selected settings On personal computer installations a software shortcut icon will be created on the desktop 2 2 Registering the MACSQuantify Software on a person
164. ta 1 0e 1 excluded 1 Oe ae from analysis g 250 moo 750 1000 1 0e 21 0e 11 0e0 1 211 022 1 063 1 0e 21 0e 11 0e01 0e1 1 022 1 0e3 FSC A A 0085 CD34 target ce 008 CD34 target ce ade 2005401 30_2062 008 1001 ae Fie sa Oegd radia 138 i e E a ee P Se cae pe adm 00S 01 30_ 2062 00 100 00 100 00 a3 t sod eg at a an PI 3 7270 7270 ess pe Ae giant 2 F 7465 5455 462 fae CD34 teget colt a2 70 MES 3650 zA 1 0 250 500 50 1000 250 500 750 1000 FSC A FSC A Figure 1 7 Gating strategy to profile CD34 cells enriched from PBMC using the MACSQuant Instrument A P1 region was defined to remove dead cells and debris B A region P2 was defined within gate P1 to select for viable CD34 cells Any remaining dead cells are positive for PI and are excluded from the region P1 P2 C The region P3 was defined within gate P1 P2 to select for all viable CD34 cells Region P3 was renamed CD34 target cells for added clarity D The final gate is displayed namely P1 P2 CD34 target cells The corresponding statistics are shown by the adjacent table E To demonstrate the gating strategy all defined regions were color coded are display on a single two parameter dot plot P1 is green P1 P2 is red P1 P2 CD34 target cells is blue The black dot plot events were excluded by region P1 For a more detailed explanation on this gating strategy download the product data sheet for MACS Control MC CD34 Stem Cell
165. taina single gate are saved the subsequent data file is smaller As a norm however it is recommended to acquire and save all data Live gating strategies can be saved for future use as Analysis templates To perform live gating 1 Click to open a new analysis window Choose the required plot design 2 Define the Experiment settings as required e g uptake volume sample name flow rate labeling strategy etc Note Ensure that the Live gate option for the appropriate gate is selected in the Experiment and Settings tab D14 Experiment Analysis template Cell Cycle odora cells Gate live P1 v C Events 10000 L 148 Note See section 4 5 4 for information about defining experiments refer to section 5 4 for information about defining experiments with multisample processing 3 Ensure that the correct instrument settings are loaded and that compensation is correctly performed Note See section 4 1 for information about performing instrument calibration refer to section 4 2 for information about performing compensation 4 Ensure that enough sample reagents and buffers are provided 5 Click on the Start Measurement button gt The MACSQuant Instrument will commence sample uptake and measurement 6 Draw regions on the plots as described in section 7 15 1 Note Click to delete all events displayed on a dot plot i e to refresh 7 Save the Analysis template for future
166. ter a project 10 Open the Reagent manager by clicking on any of the lt add gt buttons under the Autolabel tab 11 In position R1 select Universal as category and Propidium lodide Solution as reagent 12 Ensure a O incubation time and 1 99 titer 13 Click Apply and close out of reagent manager 14 Click the radio button next to PI in the Autolabel tab 15 With only position Al highlighted Enter CD133 Control in Sample ID Select Low fluidics Select Standard mode Enter 250 mL in uptake volume In Annotations tab enter Bl CD34 FITC B2 mlgG2b PE B3 PI CD14 PE Cy5 16 With only position B1 highlighted Enter CD309 Control in Sample ID Select High fluidics Select EnrichS measure Pos mode Enter 450 uL in uptake volume In Annotations tab enter Bl CD34 FITC B2 CD133 PE B3 PI CD14 PE Cy5 RI mlgG1I APC 17 With only position C1 highlighted Enter EPC Sample in Sample ID Select High fluidics Select EnrichS measure Pos Mode Enter 450 uL in uptake volume In Annotations tab enter Bl CD34 FITC B2 CD133 PE B3 PI CD14 PE Cy5 RI CD309 APC 18 Create the following display template 85 live live P1 SSC A PI PE Cy5 A 0 250 500 750 1000 0 250 500 750 1000 FSC A PE A live P14 P2 live 1000 1000 750 750 lt lt w 500 gt 500 a lt 250 250 0 0 0 250 500 750 1000 0 250 500 750 1000 FITC PE A 19 Place the Chill 5 Rack on the MACS MiniSampler wi
167. th the Reagent Rack 20 Place an open vial of PI in Reagent Rack position 1 21 Check MACSQuant Running Buffer and waste bottle fluid level 22 Check programming work by selecting View in the experimental table Acquisition Sample ID Description Flow rate Mis sample Mode Uptake volume Sample volume Annotations Autolabel a foun TC S S S o o mi fesem O S S S S o mfes i id 23 Start acquisition 86 24 Follow product data sheet for proper gating guidelines for EPC enumeration 010 04 23_2147 007 P1 ee 04 23_2147 0074 P2 ne 04 23_2147_ 0074 4P3 e 7 e P1SPAP3 lt 0 09 P1 P2 P3 P4 2 1e2 lt 7 23e2 ml lt le2 0 00 7 kd bu 0 00e0 ml a a lel lel 8 z z 0 0 af d A ome a 1 101 Jel le2 Je3 101 lel le2 Je3 101 lel le2 1e3 mlgG2b PE A CD34 FITC A FL6 APC A i 010 04 23_2147 0085P1 i 04 23_2147 0084 P2 i 04 23_2147 0084 P3 e e e PI P24 P3 D 91 11 t P14 P2 P34 P4 F m oo 2 89e1 ml E A A w lel ar ar lel Qa m m 1 8 8 1 o 0 0 1 1 10171 lel 1e2 1e3 101 lel 1e2 163 10 1 lel 1e2 1e3 CD133 2 PE A CD34 FITC A mlgG1 APC A 010 04 23_2147 009 P1 04 23_2147 009 P2 04 23_2147 009 4P3 1e3 1e3 1e3 PI P24 P3 q 81 82 PISPAP3SP4 R 1e2 ad 1e2 0 38 S q 2 26e4 ml lt e 30 W am bd 8 70e1 ml a ae ae lel ba lel f f Oo Oo 1 1 0 0 4 a 4 101 lel 1e2 1e3 101 lel 1e2 1e3 101 lel Je2 1e3 CD133 PE A CD34 FITC A
168. through the path of a laser and the deflection of light from the cell is used to provide information on physical characteristics such as size and granularity Also the laser light excites the fluorescent molecules on fluorescently labeled cells and the light emitted from each excited fluorochrome is measured by color detectors after passing through the respective filters fluorescence channels Finally the cells are discarded into the waste container 1 2 7 Displaying flow cytometric data Flow cytometry data can be displayed in four different formats by the MACSQuantify Software dot plot histogram density plot and statistic A text box can be used to enter additional information Each category is briefly discussed below however it is worth noting that data are normally visualized as one parameter histograms or two parameter dot plots Dot plot A dot plot may also be referred to as bivariant display scattergram or in some cases bitmap In this form of analysis each cell event is represented as a single dot on a two axis scale chart The position of the dot on the x y scale is dependent on the intensities of the measured parameters for that cell event Characterization of a cell population is typically achieved by displaying a dot plot where side scatter SSC y axis is plotted against forward scatter FSC x axis EBEL A 0 sa 500 750 1000 FSC A Lymphocytes Figure 1 1 A two parameter dot plot showing side sc
169. tic text box refer to Table 7 4 Tab Category Property Associated screenshot option Options View Options Header 7 Header Table Features shown using annotations v shown using annotations Table Region Regions functions Functions Description Check the header box to include this information in the text box Check the table box to include this information in the text box Column headers titles of the table can be displayed in two formats shown using annotations and shown using channel names Annotations are defined by the user The settings for regions and functions are identical to those for dot plots 133 Feature Features The recorded time scatter and function a fluorescence data acquired from each XFSCAFSCA pee k channel may be displayed using the MK FLZAFITCA ax VW FL3A PE A E ae co features setting The data shown will XC FLSAPE Cy7 A XFLEAAPCA XALTAAPCOTA be dependent on the selected regions see region functions Features can be displayed A or hidden tas by selecting or deselecting a feature using the left mouse button or by touching the display with your fingertip Cana ET Functions Statistics or Functions for each selected Feature can be displayed or hidden K by selecting or deselecting a function using the left mouse button or by touching the display with your fingertip The following statistical functions may be displayed Mea
170. to another file Conversion activate the following conversion for file export e Convert decimal point to comma the decimal point is converted to a comma e Transpose Data is exported as a table comprising rows and columns When Transpose is checked the table format is inverted i e columns become rows and rows become columns e Reverse sample order When exporting sample lists samples can be saved to an excel table in descending or ascending reverse order 3 Click Apply to implement changes Click OK close the window 116 To assign default settings for FCS export 1 Click Edit Options Software Export and FCS Files Users Experiment 4 Instrument Format Custom x Software Keyboard Lite Version FCS 3 1 x Acquire sa Export Statistic Format Best fit 16 bit Backup Regions Windows Spiona 3 Views near data Add ext info Compatible FCS OK caned _ Figure 7 25 Changing the default FCS export settings 2 Use the check boxes and or radio buttons to activate deactivate a feature e Version the default file export format can be either FCS2 0 or FCS3 0 e Format Data can be saved as Best fit 16 Bit or Float The 16 bit format is compatible with most data handling software e Options Linear All data will be saved in linear format i e without logarithmic manipulation Add ext info Information about the file format time data and file type is added to a text header of
171. to choose from Nine analysis templates are available to choose from The size and layout of the dot plot windows are predefined however users can change the properties of each individual plot using the il icon Note To modify these settings during live data acquisition the MACSQuantify Software must NOT be in analysis mode i e ail To modify Mil settings on post acquired data data opened from a file the MACSQuantify Software must NOT be in analysis mode i e Al Custom mode users can select a display format for data and text as follows 1 Click on the icon ul located beside the dot plot histogram or text table A popup menu will appear Dck do le Pisiy hd lad Hstogrsn Scar etic T Ful layer made Pru Hilir Figure 7 34 Five display formats may be chosen dot plot density plot histogram Statistic or text 126 2 Select the desired display from the popup menu ay ZUUH 12 1206s U14 nog a Censky Jo K kh Hstog am scatatic T Taxt m Fuklaver node a 50 es z n E E Procertles mao 0 25 502 750 1X0 HLA Figure 7 35 Dot plot format was chosen to display the opened dataset onan click amp 3 Optional Click to save changes to the workspace 7 14 3 Changing the properties of a plot histogram statistic or text table Note The MACSQuantify Software can display data in four discrete formats Dot plot density plot histogram and statistic In addition text may also
172. uld be used depending on the sample number and volume 5 4 1 Selecting a sample rack To select a sample rack Experiment 1 Click on the Experiment tab Sek ears 2 Select the required rack type from the Rack drop down list Rack Single tube ra rac ck 3 The corresponding rack template will popup in an independent window and will also appear in the lower section of the Experiment tab window Enoe File Edit View Mode Analysis Window Help POCE EerFRECSSNAR Oweeesa 1 2 3 4 5 6 gBOOOQ00 OC Gear Grou _ me CY OO OO OY RON RS NF NI NEA mm OPC RC CO CMC KA NI NS Nat SP mac yt pat Cpe Rate NF A Ne Nef Nf gt Tavarvrarereces Kosi DANA sok No New Figure 5 6 The Chill 5 Rack template was displayed Note The popup rack window can be closed or opened at any time using by clicking the Rack icon 5 4 2 Configuring the sample rack for an experiment Sample racks are represented graphically by the MACSQuantify Software All rack positions are given by coordinates columns are assigned numbers rows are assigned letters To select a single rack position use the left mouse button single click to activate the desired rack coordinate alternatively the MACSQuant Instrument touch screen may be used
173. ure during data acquisition live mode Reset sample on clear view plot or chart is automatically cleared after data acquisition i e it is not necessary to click the Clear icon E Export as FCS acquired data is always exported as MQD and FCS Show volume progress in uL during sample uptake and processing the MACSQuant Instrument will continually report changes to the sample volume in the Status Bar Apply express analysis The option to perform analysis in Express mode is always given 3 Click Apply to implement changes Click OK close the window 115 To assign default settings for statistic export 1 Click Edit Options Software and Export and Statistic Files Users L ACCESS i E yperiment Destination Instrument Clipboard File Remote Control Network setup F Conversion Software H Keyboard Convert decimal point to comma Timers Acquire Statistic Transpose Reverse sample order Regions ee Export sample list Options Region functions Feature functions Clipboard File Location m Public p Private Name live ls Conversions C Convert decimal point ta comma C Transpose rows and columns Reverse samples Cancel Figure 7 24 Changing the default statistic export settings 2 Use the check boxes to activate a feature Clipboard plots and data tables can be copied to Windows Clipboard File plots and data table can be exported
174. ve Files can be saved publicly in a global folder or privately in a specific user folder cap Capp bin analyses 2009 03 11 Public data organized into date folders fo data 2009 08 11 a global O device 2009 10 27 O experiments reagent analyses 2010 06 17 tT Private data data O autolabeling a organized into gt devi _ Multiparameter date or lib admin device Mutip project folders user Rebecca O experiments O service pri Tam reagent 7 3 Opening data files 1 Data files saved in the cap folder structure as outlined in section 7 2 can be opened into the Samples tab in the MACSQuantify Software Data files saved in a network location or on a USB device cannot be opened using this method 2 Open data files by e Choosing Open from the file menu e Right clicking within the Samples Tab and choosing Open e Clicking the Open icon 3 Select Data files on the left hand side of the dialog box see Figure 7 2 4 Navigate to data files to be opened 5 Data files will be displayed within the Samples tab in MACSQuantify Software r i Public al Private External Delete Workspaces Dara fies uy Data files _adm2009 12 15 2147 026 mqd Task data _adm2009 12 15 2147 026 mgd Instrument settings MC CO4_ T Cell Cocktail_h adm2003 12 19_ 2147 026 mqd KE autocamp mgd a MC_CO34_CD133_Stem_Cell_ Cocktail h Polincomp mad H La MC_CD34_ Stem_Cell_Cocktail_h er 2009 06 24_2114 011 mq
175. ve Workspaces Instrument settings Experiments Reagents and Analysis templates Analysis templates can only be saved when the Analysis mode icon Al is activated 98 2 Click on the Workspace tab Instrument settings tab Experiments tab Reagents tab or Analysis template tab to save the relevant file type Figure 7 9 Saving file types Left Workspaces Reagents file types can be saved Right Analysis mode was activated Al in order to save Analysis templates 3 Using the keyboard enter the file name 4 Click Save to save files 7 10 Importing files Analysis templates CD14 analysis Cell Cycle odora cells Instrument settings Experiments or MACSQuantify Software can import files in flow cytometry standard FCS formats MACSQuantify Software data and instrument settings can also be imported from an external file location for example a USB memory stick To import FCS files perform the following steps 1 Insert a memory stick or external media into the MACSQuant Instrument USB port Alternatively ensure that the MACSQuantify Software has intranet access to the file location 2 Click File and Import FCS file Lookin E dta o ek E 5 2009 04 28 J 2009 09 01 _Calibration My Recent CD34 Stemcell log5 Documents cD34_CD133 cocktail hlog ra cD34_CD133 cocktail log5 cD34cocktail hlog Desktop 1 17 9McC_cCD3_Pan_T_Cell_Cocktail_h mc
176. ve the closure while the container is still in the holder close it with a cap Then remove the closed container Replace the bottle with an empty waste bottle and attach the bottle closure and sensor to the new waste bottle Note Handle the full liquid waste bottle with extreme caution and dispose of as recommended by your local authority Note Itis recommended to add 100 mL of a MACS Bleach Solution to the bottom of the waste container 3 5 5 Check the instrument status The instrument status can be monitored using the status bar and illuminated bottles In order to start experiments the MACSQuant Instrument should report the status Acquisition mode tice The following procedures must be completed before performing experiments on the MACSQuant Instrument Calibration ok e Instrument hardware must be correctly installed and calibrated refer to the user manual e Instrument settings must be correctly calibrated and compensated refer to the user manual A more comprehensive explanation on monitoring the instrument status is given below Checking the instrument status using the status bar The instrument status is reported by the status bar using text and a corresponding color code Orange MACSQuant Instrument in Data analysis mode the instrument can only analyze data The instrument must be placed in acquisition mode to perform a measurement Data analysis mode Time Volume Rate Count 6 gt WAS 7
177. warranty from their manufacturer Miltenyi Biotec must be informed immediately if a claim is made under such warranty If a material or manufacturing defect occurs within the warranty period Miltenyi Biotec will take the appropriate steps to restore the full usability of your Product Limitation on damages Miltenyi Biotec shall not be liable for any incidental or consequential damages for breach of any express or implied warranty or condition on this Product Some states or jurisdictions do not allow the exclusion or limitation of incidental or consequential damages so the above limitations or exclusions may not apply to you This warranty statement gives you specific legal rights and you may have other rights which may vary from county to country or jurisdiction to jurisdiction 185 Air filter Air filter connector APC MACSQuant Column MACSQuant Instrument MACSQuant Running Buffer MACSQuant Storage Solution MACSQuant Washing Solution Bottle closure Column connector 15 Glossary Hydrophobic 0 2 um air filter attached to the bottle closure Used to vent the bottle and simultaneously prevent contaminants from entering or escaping from the fluid bottle Luer to thread connector for attaching the air filter to the threaded bottle closure vent Allophycocyanin Separation column specifically designed for the MACSQuant Instrument An automated flow cytometer ACSQuant Analyzer MACSQuant VYB or MA
178. wn can only be performed by Custom users and administrators 178 1 Click Edit Options Software and Timers Options Users Experiment E Instrument Software Keyboard Standby timer 120 min Acquire Shutdown timer 5 min Export Rien Need t Backup eedle priming time 0 min H Regions Views Timers Shutdown default behavior Analyse mode Instrument off Figure 9 1 Timers window 2 Click on the Shutdown time field Modify the time minutes using the slider bar or keyboard 3 Click Apply and OK 179 10 MACSQuant Live support MACSQuant Live support is a real time diagnostic service provided by Miltenyi Biotec Technical Support Highly trained MACSQuant Experts can be reached in real time to assist with any queries you may have Note As an option is it possible to use a web cam in communication with MACSQuant Technical Support If no web cam is provided please contact your nearest MACSQuant Specialist Note The MACSQuant Instrument must have network access to the internet for live support Contact your local IT administrator if this is not the case To receive remote assistance 1 Select the Tools menu 2 Click MACSQuant live support A popup HTML field will appear Complete the fields with your information and detail any queries you may have using the Message Question box E HTML Help To talk with a representative please answer the following
179. xample using an analysis template Lon The Single tube rack Chill 5 15 or 50 Racks and 96 well formats can be Single tube rack Chill 5 tube rack H _ H Sele ey chosen using the Rack drop down list Chill 50 tube rack 96 well Filename NS2009 01 28 001 mqd The filename is shown is this field not changeable The filename is user s initials and date followed by file number SempeD ____ Sample ID can be entered using this text field Description _____ Sample description can be entered using this text field Selecting Analysis from the Mode drop down list reveals a list of analysis templates available for immediate use These templates correspond to the MACS Control Cocktails and count programs For more information on the MACS Control Cocktails please refer to www miltenyibiotec com MC_CD14_h v Selecting Setup from the Mode drop down list reveals three options for instrument setup Calibration Compensation and Compensation 7Colors Calibration v n NOTE Setup is only available to administrators and Custom users Compensation Colors Initials of user in the top left corner in this example Express User EU 156 Folder icon to open Workspaces Instrument Settings Experiments Analysis templates and or Data files depending on user access rights set by the administrator Click to save Workspaces Instrument settings Experiments and Analysis templates depending on user access rights set by
180. xperiments or Data files may by opened by Express mode users Custom mode users and administrators are able to open Workspaces Instrument settings and Analysis templates in custom mode 166 2 Click on the Experiment tab or Data file tab to open an experiment definition or data files respectively Figure 8 9 Highlighted Experiment tab to open the desired file type To open experiment definitions 1 Highlight the Experiment tab on the Open window 2 Highlight the file location Private Public or External 3 Select the file type and click Open e To open data files 1 Highlight the Data files tab on the Open window 2 Highlight the file location Private Public or External 3 Select the file type and click Open Le 8 6 3 Saving files 1 sais to open the Save window 2 Click on the Experiment tab to save the relevant file type Figure 8 10 Highlighted Experiments to save the desired file type Users are able to save experiment descriptions 167 To save a workspace 1 Highlight the Workspace tab on the Save window 2 Highlight the desired file location Private Public or External 3 By default the workspace will be saved to the user s private folder To save to an external drive highlight the External tab Esna Note If no external media is attached to the MACSQuant Instrument or personal computer USB port the follow error will be reported Ext
181. y selecting it from the drop down menu under the button Set up to visualize the first fluorochrome requiring compensation e g FITC in the histogram In the Experiment tab program all necessary parameters such as Sample ID and uptake volume Open the 8x8 7x7 compensation matrix but clicking the instrument settings icon and selecting the compensation tab Draw a scatter gate around the population of interest within the FSCvSSC dot plot This will be P1 10 Display only events in P1 in the histogram plot by selecting P1 from the drop down menu of the plot header 11 Draw an interval around the positive P2 and negative P3 populations Optional Rename P2 and P3 regions to positive and negative respectively by right clicking on the region and selecting region properties 12 In the statistics table format properties under the i button Select to display P2 and P3 regions under the region functions Select to display the median of all the spill over fluorescence channels e g for FITC spill over display V1 V2 B2 B3 B4 R1 and R2 although not all require compensation 13 Enter values in the box within the compensation matrix by using the associated slider For example compensating FITC signal from the PE detection channel the fourth box down within the third column should be adjusted Once the value has been entered into the matrix select the file to be recompensated from the sample list 54 14 R

MACSQuantify™ Software guide Basics of flow cytometric analysis

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MACSQuantify&trade; Software guide Basics of flow cytometric analysis

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MACSQuantify™ Software guide Basics of flow cytometric analysis