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Labcyte Echo Dose Response Application

1. Re Survey every source plate C Maintain survey history Output Options Output Files f Run specific Source Plate specific affected by processing order Transfer Reports Save transfer reports as file type M xML M csv TXT Format lt DEFAUL T gt Directory lt DEFAULT Filename DEFAULT Survey Reports Save survey reports as file type M xML M csv TXT lt DEFAULT gt Directory lt DEFAULT gt Filename DEFAULT Figure 4 24 Report Format Options Echo Dose Response User Manual 83 Software Reference The Output Format Builder window shows all the report variables left selection box that can be added to the report format right selection boxes The list of variables changes with the report area that you select Output Format Builder Figure 4 25 Transfer Output Format Builder 84 Report Directory The default report directory tor both surveys and transfers is C Labcyte Echo Reports You can specify a different directory by clicking the Directory button and entering a new location Use the Select Folder button to browse to a different directory Folder Name Build er Form Figure 4 26 Folder Name Window Report Filename The Echo Dose Response software uses the following default formats to report the survey and transfer results Filename Builder Form Figure 4 27 Transfer Report File
2. ooooonccicnnoccconoonccononnncnnonos 97 Preface Intended Audience The Echo Dose Response program is designed for use by laboratory personnel who are familiar with preparing microplates for IC50 analyses This software application automates sample transfer and multiple dilution process to produce the final assay microplates Software Requirements To use the Echo Dose Response application the following items are required e Echo 5XX Liquid Handler system using software version 2 1 or later e License key trom Labcyte Inc e Computer system meeting the following requirements Operating system Microsoft Windows XP SP2 or later Net Framework 2 0 CPU Intel Pentium 4 or later 2GHz or greater Memory 512 MB or higher Network connection 10 100 1000 BaseT Network protocol TCP IP Hard drive storage 200 MB Video 1024 X 768 resolution 256 colors or better The Echo Dose Response application works closely with the Echo 55X client software and should be installed on the same client PC Manual Organization This manual is provided on CD and accompanies the Echo Dose Response installation CDs Software procedures and screen descriptions in this manual are also available in the Help section of the Echo Dose Response application The Echo Dose Response user manual is organized as follows Chapter 1 Introduction Chapter 2 Getting Started Chapter 3 Software Operation Chapter 4 Software Reference Appendix Sample
3. E tte Chasin He amare plate jampisis Beki ha aka in a maapinda eral PaA tiik on one H e burni n Ba Legend e etna tha fpa for ma prinia rada in chet eta pia spia Teen hj i on PiE Eaa Pai Troe ERED A 00 0 ea Edi dee e e e e e e e ee dd o ds dle ds dl dl i de de dedo T TPE ee ee 4444444 4444445 eo a Dn EE ee gg O E a EN a E o Ly a ES a a E a i AAA AA Ae de e e eo e rd dde de AAA ete ale de de e a e i eg ee i rd dedo dedo ha de do dd b eh eo oo oe cae de e e e e e e de e e e P oo e OO HO 4 OF te e de e de de e de e o e a ee a a A ENNS AR de 4 ES eo oS Pe di www labcyte com 1190 Borregas Avenue Sunnyvale CA 94089 Tel 877 742 6548 Tel 1 408 747 2000 Echo Dose Response User Manual Labcyte Part Number 001 5722 Revision 2 December 2007 The information contained in this manual is subject to change without notice Any unauthorized changes or modifications to the Echo Dose Response application will void its warranty Contact Labcyte Inc prior to making any change or modification Copyright 2007 Labcyte Inc All rights reserved Printed in the United States of America Microsoft Windows Windows XP ActiveX and Microsoft SQL Server are either registered trademarks or trademarks of Microsoft Corporation in the United States and or othe
4. A00 9090 OOOO OOO OOOO 90000 O 1900 OOO 00 OOXOXIXOOOOOOOOOOD00O 219409 OOOO OODVOOOODOD0DO Bit Seeeeeeeeeeeeeeseee 94800 eeeeeeeeeeeeeseees Oil Seeeeeeeeseeeeceeee OOOO D 40000000090000099 9 9 0 E 4040000000000000099 9 9 09 y ISSO SOS Figure 4 8 Source Plate Designer Step 3 Echo Dose Response User Manual 61 Software Reference 4 2 3 Destination Plate Designer The Add Destination button begins the Destination Plate Designer wizard in a new window This wizard helps you prepare a template of tinal compound concentrations that will be used to generate a dose response curve Step 1 Design the Destination Plate window Defines the destination plate type Assigns wells to DMSO or blocks wells trom compound transfer See Step 1 Fields and Buttons below Destination Plate Designer E 2j x Step 1 Design the destination plate template Select the wells in the microplate and then click on one of the buttons in the Legend to define the type for the selected wells in the plate template When finished click on Next Destination Piate Type y theists is Le 7 elo pops fra jas feliz re fro feo far fzofasfar ME CX E m0 e C Avaicbie gt O O Q O O O O O O O O O O O O O O O O 000000000000000 O 00000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 0
5. 0790 Survey Intermediate Dilutions Plate 010 03 16 KB Microsoft Office Excel 1 2 2007 6 57 PM ES5x 0790_ Survey Intermediate Dilutions Plate 010 xmi 1J44KB XML Document 12 2007 6 57 PM E ES5x 0790 Survey Source Plate csv 1KB Microsoft Office Excel 1 2 2007 6 57 PM Figure 3 40 Transfer report directory From this directory location you can retrieve the following reports 4 Run ID 1168130535 Run Date Time 200 7 Jan 06 04 42 15PM 3 Application Name Labcyte Echo Dose Response Application Version 1 0 0 5 Protocol Name 16 compound assay edr ea p y 6 User Name jsmith DETAILS source Survey Survey Plate Source Fluid Fluid Survey Source Plate Name Barcode Source Plate Type Well Height Volume DMSO Status Source Plate Labcyte 384PP StdV At 2 364 36 629 85 Source Plate Source Plate Run ID 1 2E 09 Source Plate Run Date Time 2007 Jan 06 04 42 15PM 14 Source Plate 3 Application Name Labcyte Echo Dose Response Source Plate 4 Application Version 1 0 0 Source Plate Protocol Name 16 compound assay edr Source Plate 6 User Name jsmith Source Plate Source Plate 8 DETAILS source Plate a Source Plate Sein di aney Aey 22 Source Plate Plate Source Fluid Fluid Survey 23 Source Plate Source Plate Name Barcode Source Plate Type Well Height Volume DMSO Status 24 Source Plate Intermediate Dilutions Plate 01 Labcyte 304PP StdV 41 2 364 36 629 05 25 Source Plate Intermediate Dilutions Plate 01 Labcyte 3
6. 42 32PM Please insert Source Plate Labcyte_384PP_StdV 06 Jan 2007 04 42 43PM Please insert Intermediate Dilutions Plate 01 Figure 3 36 Transfer protocol in progress 4 Click the Start button gt to begin the sample protocol The software begins by displaying prompts to remove plates currently in the Echo liquid handler 5 Click OK in the plate prompts after you remove any existing plates from the Echo liquid handler Echo Dose Response User Manual 45 Software Operation 46 The first plate prompt for the sample protocol is to insert the source plate Plate Prompt i ajaj Ploasc insori cource plato Pate Name Source Piste Pete Type Labcyte_364PP_SdV Fate led SOURCE PLATE 42 10 MH INSET Figure 3 37 Source plate prompt during transfer run The prompt box displays the plate template name plate type plate insert and a field for the barcode or plate ID When you are using multiple source intermediate or destination plates the barcode or plate ID field provides an additional check Verify Barcode to identify the correct plate The software will compare the barcode you enter to the plate that is inserted in the Echo liquid handler and scanned by the barcode reader If the barcode doesn t match or if you enter the same barcode for two different plates then the software will display an error message If you use plate IDs the software will check for inconsistencies for exampl
7. Number of points needed for IC50 graph 12 point curve Set up the transfer protocol Use the same procedure described in Example 1 to set up the protocol for Example 2 For this example run 2 replicates Run the Protocol 1 Open the Aun Options screen 2 Select the Full plates option if it is not already selected 3 Enter 4 for the Number of Full Source Plates 2x Instrument JECHO2 Serial Number E55X 0790 Status Available Normal Protocol Name C Labcyte Echo Protocols 12 gkw m Run Protocol Full plates as defined in protocol Number of Full Source Plates 4 gt Partial plates as defined by a pick list Simulate Figure 4 32 Entering number of source plates Echo Dose Response User Manual 97 Software Reference 4 Click Simulate to open the simulation window 5 Click Play to start the simulation Since this protocol uses several plates it requires extra time to load the plate data and start the simulation 12 pt w mult src edr Simulator 12 ptw mult src edr Source Plate Type Protocol Jlabcyte_384PP_StdV Source Plates i Ee ce ae eae Fees G CILLIT rta Hatan meee O2S020899090959000 ARA Cege DOOCOOOGOLLOCLOOOO di eee 9O280000090005050 pears Eeee 9900000009 0900000 Dese O0C00080005005055 A 5 1009 400000 0V0V0O0CI DODO COG OOOCOOOOOCLCOCCOQDO ment Transfer
8. Q Torop 0 20000000 0000000099 8800000000000000 EDE SSC COCOCCCOCOCCCCOCCCECS E 1000000000000000 0000008 4 MAO SOCOCCOCOSCCCCECOCCCECES TOCO OC OOOO L OOOO OOOE COCO OSCOCCOSCCCOCOCSOCOOCCCS nO OOOO OO OOSOOXOOIOO OOOO OOOODOOODO OD OOOODO O OOOO Figure 3 8 Replicate compounds EN 90 KOG 00 MOO D 5 Assign controls and or DMSO To enter controls a Select the column or row to assign to controls b Click the Control button c Enter the control name in the prompt box and click OK The control wells are highlighted in blue Figure 3 9 Add controls To enter DMSO wells a Select the column or row to assign to DMSO b Click the DMSO button The DMSO wells are highlighted in yellow r Well Selection Actions Compound Conc E Control i Reserved Available Figure 3 10 Add DMSO HINT You can include controls and DMSO in the compound replicating pattern Assign individual wells to controls and DMSO Select the compounds controls and DMSO Enter the compound name starting number direction and then click Replicate Pattern Only the compounds will be named and numbered 6 Click the Next Button and enter the source template name Type a name in the Template Name field or click the left arrow lt lt button to accept the name that is suggested Source Plate Designer Step 2 Choose a name for the source plate template The name of the template must
9. and change the offline backfill volume to 50 nL Automatic DMSO Backfill I Perform Backfill First f Last Maximum Backfill Volume 75 0 nL W Backfill offline for volumes greater than equal to Yellow denotes offline backfill Figure A 11 Dose response curve box The data table automatically highlights in yellow the backfill volumes gt 50 uL that will be filled offline Source ie Backfill Volume Concentration eee inl Volume Assay ul a a a soe al 25 gt gt e es lf o a a a esco lf 25 a senor sl 70 gt a gt gt a a Figure A 12 Dose response data table showing offline bulk fill g Enter the following information in the Aeo icates box right side Enter Number of Replicates 3 Select the Same Plate and In Adjacent Wells options Select the Curve Fill Direction Across Select the Compound Fill Direction Down h Click the Next button to enter a destination template name I Click Finish You are done with the destination plate template To preview or edit this template from the Plate Design window click Destination Plate in the protocol tree Click Edit to enter the template designer Echo Dose Response User Manual Appendix Plate Design Options Name 12point curve Description Uses 5 mM source concentration with final assay volume set to 20uL This example incorporates the controls and DMSO
10. be unique within the system The plate preview of the plate is also shown below Figure 3 11 Source plate template name Echo Dose Response User Manual 25 26 Software Operation 7 Click the Finish button to return to the Plate Design window The source plate definition is complete The Pate Design window displays the design of the source plate in the left pane Protocol 1 Plate Design Options Name 12 point 5 4840g curve Description Uses 5 mM source concentration with final assay volume set to 20 uL This example incorporates the controls and DMSO backfill in the destination plate Plate templates in this protocol m Plate Template Actions Add Intermediate Add Destination There are 2 source concentrations per compound in this protocol m Process plates in this protocol e Automatic Protocol defines the optimum sequence Source plate centric Minimize source plate swapping Destination plate centric Minimize destination plate swapping Figure 3 12 Source plate definition completed If you need to edit or delete the source plate template you can select the source plate template in the protocol tree and click Edit to open the Source Plate Designer window or Delete to remove the source plate Plate templates in this protocol Protocol Plate Template Actions Add Source Add Intermediate Add Destination Figure 3 13 Source plate te
11. bulk fill direction column or row d Select the wells that will be bulk filled with DMSO Note You can only select in the direction that is specified in the Bulk Fill Direction box Echo Dose Response User Manual 91 Appendix 92 Click Next to continue to Step 2 Enter the number of intermediate concentrations you want to create In this example enter 2 Two concentration fields will be displayed Select the compound concentrations from the drop down menu Enter the transfer volume for the first concentration In this example enter 2 5 nL Enter the volume of DMSO you want to bulk fill 25 pL The software will calculate the final concentration of the compound in the intermediate plate Repeat steps e to g for the second intermediate concentration 250 nL transfer volume 25 pL bulk fill Intermediate transfers How many intermediate concentrations would you like 2 Source Concentration Transfer Volume Bulk Fill Volume New Concentration 5 000 03 M 5 mL 25 00 uL 5 000E 07 M 5 000E 03 M 250 0 nL 25 00 uL 4 950E 05 M Figure 4 30 Determine compound concentrations The intermediate plate template reserves two wells for each compound concentration Click Next to enter an intermediate template name Click the Finish button to return to the Plate Design window The intermediate template is done To preview or edit this template from the Pate Design window click Intermediate
12. by reducing or eliminating the need for pipettor tips reservoirs and other labware required for compound dilution and assay plate preparation 1 1 What is IC502 IC50 is the concentration of a compound that causes a 50 inhibition of enzyme activity in vitro This measurement is commonly used as a measure of effectiveness The smaller the amount needed the better the compound is as a drug lead Another measure of drug efficacy is EC which represents the concentration of a compound that is required to obtain 50 of the maximum effect in vivo Other measurements are LD50 and TD50 briefly described below IC50 is how much is needed to inhibit 50 LD50 is how much is needed to kill 50 Lethal Dose EC50 is how much is needed to cause a 50 effect TD50 is how much is needed to cause a toxic effect Therapeutic window is the distance between IC50 and TD50 See Atto www taa qgovwohrms aockets ac 00 slides 362 15 1d sld036 Atm Echo Dose Response User Manual 1 Introduction 1 2 What Is a dose response curve A dose response curve is a graph that measures the amount of a drug or toxin given to the response of the organism to that drug The midpoint of the curve represents 50 inhibition 90 activity ra logE CS0 log Cancentration 1 3 How does the Echo Dose Response software fit in The process of screening a compound for potential drug development includes the following steps 1 Test potential c
13. following steps Click the Save As button to open the Save dialog box b Browse to the folder that will store the file The default folder is C Labcyte Echo Protocols c Name the file select csv as the file type and click Save d Open the csv file in a spreadsheet program EY DMSO_fill_int csv C E Name Intermediate Dilutions Plate with DMSO 01 Type Labcyte 384PP StdV 3 Wells 384 Fluid DMSO 5 Fill Column 6 7 Column Volume Units 1 30 uL EN 2 30 uL 3 30 uL 11 4 30 uL Figure 3 39 Bulk fill data in a spreadsheet At any time during the protocol run you can pause or stop it When the protocol run is done the progress bar will show 100 complete and the Close button will become active Echo Dose Response User Manual 47 Software Operation 3 7 Manage Transfer Data At the end of the transfer run the Echo Dose Response software builds the transfer reports and stores them in dated directories in the following location C Labcyte Echo Reports Labcyte Echo Dose Response dl Hamne Date Modified 1167789741 Intermediate Dilutions Plate 01 Bukral 1KB BULKFILL File 12 2007 6 54 PM El E55x 0790_Prnt_lis7792868 csv 2 Microsoft Office Excel 12 2007 6 57 PM ES5 0790 Print_1157792868 xml 10 KE XML Document 12 2007 6 57 PM El ES5 0790_Prmt_115779 2879 c5w BKB Microsoft Office Excel 1 2 2007 6 57 PM ES5 0790 Print_1is7792879 xml 39KB XML Document 12 2007 6 57 PM al E55x
14. is programmed with several compatible destination plates however you can add destination plates through the Echo Dose Response software or the Echo liquid handler software See Labware on page 73 for more information on plate definition See online Help for procedures on adding editing or deleting labware Echo Dose Response User Manual 75 Software Reference Fields Name User defined name to identify the plate This name will be listed in the Plate Type List window 2 x Connected To ECHOZ Cache Plate List Localhy Labcyte_384PP_StdW Labcyte P 05525 Source Greiner_384P5_ 781096 Greiner 781096 Destination Labcyte_1536C0C_LoBase_Dest Labcyte P 05850 Destination Labcyte 384C00C_ LW Dest Labcyte P 05710 Destination Labcyte_384PP_StdW_Dest Labcyte P 05525 Destination Labcyte LDV 384 Dest Labcyte LP 0200 Destination Lopy Aurora_3456C0C_ 00017829 Aurora Discovery 00017829 Destination Greiner_1536HiBasePS_782096 Greiner 782096 Destination Delete Coming_1536COC_HiBase_Dest Coming F 07200 Destination Edit Import Barcode Location Location of barcode label on the destination plate None Left Right Long Manufacturer Name of the plate manufacturer Lot Number Lot number assigned by the plate manufacturer Part Number Part number assigned by the plate manufacturer Rows Number of rows in the microplate Columns Number of columns in the microplate Al XOffset A Distance from left outside edge to center of first col
15. lt P XAXKXJ A JAI vatate XX seecececesesesesceeseseeeeeseseeeeeeeeee Available 2 2e 2 ented OOOO Replicate Pattem Select the wells to copy Enter the base compound name and starting number click the direction to copy the wells and then click the button below SARN 00 00 OE OE IN Base Name y Starting Number 1 Replicate Selected Pattem Ez Undo Replicate Pattem A a ee IN 4 Lt nS 0 SS X A A SOS SOS Across Down Figure 3 6 Unused wells blocked b Select the wells you want to replicate The selected wells represent the pattern that will be replicated and numbered c Enter the base name for the compound starting number direction to number the compounds across or down and then click Replicate Selected Pattern Replicate Pattem Select the wells to copy Enter the base compound name and starting number click the direction to copy the wells and then click the button below Base Name Kinase Starting Number Across C Down Undo Replicate Pattem Rename Selection Figure 3 7 Compound name and starting number Echo Dose Response User Manual 23 Software Operation 24 The software will replicate and number the compounds in the direction you specified TERR M OOCODDDODOOODODOOOOOOODOOOG Ds Seeseseccscsscecsscees 5 000E 04M Kinas O
16. need to complete the processing of the destination plate and put it in environmentally controlled plate storage Process plates in this protocol Automatic Protocol defines the optimum sequence Source plate centric Minimize source plate swapping Destination plate centric Minimize destination plate swapping Figure A 13 Process plate options Hint You can save processing time by turning on the Survey History function This function saves survey data for the source plate and reuses it the next time that the source plate is returned to the Echo liquid handler For more information go to Survey History on page 80 7 Check the protocol for missing or inconsistent data Select Error Check from the Protoco menu The transfer protocol is done Test the protocol in the simulation program before using it 96 Example 2 12 pt curve using multiple source plates Plan Curve Data This example is the same 12 pt curve protocol that was used in Example 1 however it uses multiple source plates Starting concentrations of your compounds Layout for the source plate One source plate template containing compounds 4 source plates specified in Run Options controls and DMSO Layout for the intermediate plate One intermediate plate with two dilutions from stock 1 100 250 nL 25 uL 1 10 000 250 nL 25 uL Layout for the destination plate 12 pt curve 2 controls 2 replicates Final assay volume 25 uL
17. the color that matches the Well Color Legend Well Color Legend Compound EE Control _ DMSO The Protoco intormation box upper right provides details about the transfer protocol such as the number of plates that will be needed As the simulation proceeds this box is updated to show which plates are currently in use Protocol CLabcyte Echo Protocols 5 point dose source Plates 1 Destination Plates 5 A Sessions 1 Current Session 1 Current Transter Map 1 Transfer trom Source Plate 1 1 Transfer to Destination Plate 03 4 4 4 Labware The Echo Dose Response software relies on the labware definitions resident in the Echo liquid handler ae Preferences database You can add new plate definitions or manage the existing ones by selecting Labware Labware Definitions Definitions from the Zoo s menu eee 4 4 1 Plate Type List The Plate Type List opens to display the source and destination plates that are available from the Echo liquid handler database If you are not connected to an Echo instrument the list is cached locally stored in the Echo Dose Response software iS Plate Type List x Connected To ECHOZ Cache Plate List Locally Manufacturer Labcyte_384PP_StdW Labcyte P 05525 Source Greiner 384P5 781096 Greiner 781096 Destination Labeyte_1536C0C_LoBase Dest Laboyte P 05850 Destination Labcyte_384C0OC_ LW Dest Labcyte PF 5710 Destination Labcyte 384PP StdW Dest Labcyte P
18. transfer to or from selected wells Assia By default all well positions are available Use this button to deselect well positions that have been previously selected Selects all wells in the plate template for quick well assignment Same action as clicking the corner cell above well A inca J Unselects all wells in the plate template that were previously selected Bulk Fill Direction specifies the direction that the assay wells are to be filled by a bulk tiller Note The selected wells are used as reservoirs for DMSO only During plate preparation or during the protocol run when the bulk fill screen is displayed the selected wells are filled with DMSO either manually or with a bulk filler The Echo system uses the DMSO in these wells to backtill the compound wells in the assay plate Echo Dose Response User Manual 59 Software Reference Step 2 Build Intermediate Plate Specifies the number of intermediate transfers and calculates the compound concentrations See Step 2 Fields Intermediate Plate Designer y 2 xi Step 2 Build Intermediate Plate Select the number of intermediate concentrations and fill out the table below Choose the transfer volume and select the bulk fill volume per new concentration Click on Next when finished Intermediate transfers How many intermediate concentrations would you like 13 0 005 M y 150 0 nL 30 00 uL 2 488E 05 M Figu
19. 00000000000000 000000000000000 000000000000000 000000000000000 000000000000000 A O O Figure 4 9 Destination Plate Designer Step 1 Fields and Buttons Source Plate Type Drop down menu of available destination plates Irons 245COC 00017829 Greiner_1536HibaseP5_ 82096 Plate map of the selected destination plate Greiner_384P3_781096 e Coming_1536COC HiBase Dest Well positions that can be clicked and assigned to the Labeyte_1536COC_LoBase Dest components in the We Selection Action box pot ae 62 Well Selection Action Components that can be assigned to the selected well positions C DMSO Assigns wells to hold DMSO for bulk filling compound wells in the intermediate plate or assay wells in the destination plate NT Prevents liquid transter to or from the selected wells Reserved C Pare By detault all well positions are available Use this Available o button to deselect well positions that have been previously selected Selects all wells in the plate template for quick well assignment Same action as clicking the corner cell above well A Unselects all wells in the plate template that were previously selected Step 2 Define the Dose Response Data Points window Selects the number of intermediate concentrations This number corresponds to the concentrations fields in the table See Step 2 Fields Destination Plate Designer i 2 x Step 2 Define the dose response data points Sel
20. 05525 Destination Labcyte_ LDV 384 Dest Laboyte LP 0200 Destination Aurora 3456C0C 0001 78 29 Aurora Discovery 00017329 Destination Gareiner_1536HiBasePS 782096 Greiner 782096 Destination Coming _ 1536C0C_HiBase_ Dest Coming F 07800 Destination Figure 4 17 Plate Type List Note If you have installed the Echo Dose Response software and receive an error message followed by a blank Plate Type List you will need to connect to an Echo instrument and cache the plate list from the Echo instrument Echo Dose Response User Manual 73 74 Software Reference Command Buttons Cache Plate List Locally Click this button to save the plate list trom the Echo liquid handler to the Echo Dose Response software Add a plate definition Only a destination plate can be added Refer to one of the following options Add a labware definition trom the Echo Dose Response software Add a plate definition from the Echo liquid handler software and reload the updated labware definition to the Echo Dose Response software Edit a plate definition Select a plate definition and click the Edit button Source plate definition Only the barcode location of the source plate can be edited Destination plate definition Edit any setting in the definition Copy a plate definition Select a plate definition and click the Copy button You can copy a source or destination plate definition to create a destination plate definition Delete a plate definitio
21. 1 control Select the option Include Controls in Dropdown Lists Dose Response Curve Number of data contral points per curve 13 Include Controls in Dropdown Lists M Figure 3 18 Number of data points per curve The Data Point Curve table will automatically update to display the number of rows corresponding to the number of points in the dose response curve 30 This example appends a control to each curve instead of using separate columns in the plate 5 Enter the following data in the Data Point Curve table see Figure 3 19 Select the compound concentration from the drop down menus Data points 1 4 5 00E 03 Data points 5 8 4 95E 05 Data points 9 12 5 00E 07 Control name 13 Positive Enter the transfer volume for each data point Data points 1 4 75 25 7 5 2 5 nb Data points 5 8 75 25 7 5 2 5 nb Data points 9 12 75 25 7 5 2 5 nb Control volume 75 nL 5 000E 04 D CE s aseos ell o aseo 250 ES o IPEE IRE k CAE E E Figure 3 19 Dose response data point table 6 Return to the Dose Response Curve box see Figure 3 19 Enter the Assay Volume added to all destination wells 25 00 pL Select the option Automatic DMSO Backfill Select the timing for the backfill Last after compound transfer Echo Dose Response User Manual 31 Software Operation Enter the maximum backfill volume 75 nL The maximum backfill volume ena
22. 50 Calculator The Echo Dose Response Calculator can be found in the Echo Dose Response directory See previous Figure 2 8 You will need Microsoft Excel to open this document Echo Dose Response User Manual 9 Getting Started 2 3 Main Window When you open a new or existing protocol the Main Window displays the Plate Design window in the left pane and workflow diagram in the right pane The workflow diagram serves as a guideline to setting up the microplates in the protocol E Labcyte Echo Dose Response TIE Toolbar j File Protocol Tools Help DEHE D Protocol p 384 well plate assay edr Navigation and Exit buttons gt gt File Name Plate Design Options Name Deseiption Smilin 12408 PP source plate Echo prepares intermediate concentraban 25 uM to marcos a Wheont cure foreach compound Each 324 well destination plate holds 32 CURS piua postive and pagaba controls Total assay volume iz 50 ul Plate Design Intermediate htermedate Ddutions Plate wih DMSO Window E Destination Destination Plate There are 2 source concentrations per compound in thes protocol p Process plates in this protocol 2227727272 Workflow and Automatic Protocol defines the optimum sequence t m t r vi w Source plate centrc Minimize source pate swapping e pla ep evie Destination plate certic Minimize destination plate swapping Status gt comected o ECHO
23. 555 s n Pessx 0790 Ver 2 1 4 Bar 10 Figure 2 10 Main Window Screen Components The components in the Main Window are described below Toolbar The toolbar contains software menus with functions to open and save transfer protocols set up the software define labware run the protocol customize protocol options and display the online Help The cons in the toolbar provide shortcuts to the most frequently used functions such as the Aun Protoco tunction For a detailed description see Joo bar on page 65 Protocol file name The protocol file name identities your transfer protocol in the computer s directory The software uses an edr file extension and stores the file in the default directory C Labcyte Echo Protocols You can store your protocols in different directories Navigation and Exit buttons The left and right arrow heads 4 t become active when you have too many protocols open to view all of the file names The arrows shift the protocol tabs left or right to display the file names The Exit icon x closes the displayed protocol Plate Design window The Pate Design window contains all of the required protocol data necessary to perform the compound transfer For a detailed description see Plate Design on page 51 Options window The Options window behind the Plate Design window displays protocol specific options such as survey history output file type and report formats For a
24. 64PP StdV AZ 2 364 36 629 05 26 Intermediate Dilutions Plate 01 Labcyte 364PP StdV A3 2 364 36 629 05 ad Maa Intermediate Dilutions Plate 01 Labcyte 304PP StdW 44 2 464 36 629 05 a Model Intermediate Dilutions Plate 01 Labeyte 364PP StdV AS 2 364 36 629 05 E Intermediate Dilutions Plate 01 Labcyte_ 364PP StdV AG 2 364 36 629 85 Intermediate Dilutions Plate 01 Labcyte_354PP_ StdV A 2 364 36 629 05 17 Intermediate Dilutions Plate 01 Labcyte 384PP StdW AS 2 364 36 629 05 Figure 3 41 Intermediate and source plate survey reports in csv format 48 Ses ee EA FEAR ae ee eee Al Run ID 1168130535 Run Date Time 2007 Jan 06 04 42 15PM 3 Application Name Labcyte Echo Dose Response 4 Application Version 1 0 0 Protocol Name 16 compound assay edr 6 User Name jsmith DETAILS Source Source Destination Plate Source Source Concentration Plate Source Plate Name Barcode Well Concentration Units Destination Plate Name Barcode source Plate Ad 0 005 M Intermediate Dilutions Plate 01 Source Plate Ad 0 005 M Intermediate Dilutions Plate 01 source Plate B1 0 005 MM Intermediate Dilutions Plate 01 Source Plate B1 0 005 MM Intermediate Dilutions Plate 01 Source Plate C1 0 005 M Intermediate Dilutions Plate 01 Source Plate C1 0 005 MM Intermediate Dilutions Plate 01 source Plate D1 0 005 M Intermediate Dilutions Plate 01 47 Source Plate D1 0 005 M Intermediate Dilutions Plate 01 18 Source Plate E1 0 005 M Intermed
25. 7 eHO7 cDimtgEc303gq0 AeGOQlpHexsnBketwW22F3o3 EGqjoTmo 8078y36FEJMBA5c3jVpi52ZB3j7IvxBUuo S7MZ2cRInECETCOVQbmbthirsLSNzk1o0 gelvHFP oNxTHIOCSE qe DAA Leg iD CEN 5 YMNCTU fo KEY END Activate Cancel Figure 2 12 Activating instrument Note The Get Current Key function is a useful troubleshooting tool to verity that the key is written correctly to the instrument When you click the Get Current Key button the software will search for the license key and insert it into the cense Key text box Send the encrypted key to Labcyte to determine if the license key is corrupted or if it applies to a different instrument 5 Click the Activate button The Echo liquid handler is now activated You will not have to perform this procedure again unless you need to activate a different instrument Echo Dose Response User Manual 13 Getting Started 2 4 2 Setup preferences Your laboratory applications for dose response analyses may require the transfer data in a specific file and data format The Echo Dose Response software provides transfer data in three file formats csv xml and txt with reporting options that can be customized to meet your needs 1 Open the 7oo s menu and select Tools Help Preferences Labcyte Echo Dose Response Defaults i 2 x Options Protocol Options Surveys f Re Survey every source plate Maintain survey history Output Options Output Files f Run specific Plate s
26. Compound 16 is at P1 Compound 240 is at P15 and is the last compound well because the remaining wells have been reserved EUR Miri 554 4 2999999999999999 5 000E 03 M Compound 16 2 m Unselect All Figure A 4 Replication completed Select the reserved wells that will contain the first control columns 16 17 Click the Control button Type the control name in the prompt box Positive Click OK DIRA 90800000000000 Control Positive Cn i r Figure A 5 Assign control wells Repeat for the negative control in columns 18 and 19 The controls are labeled a and b on the plate map Select the remaining wells in the plate and click the DMSO button Figure A 6 Control and DMSO wells assigned Enter a new source template name in the Template Name field or click the left arrow lt button to accept the suggested name q Click Finish You are done with the source plate template To preview or edit this template from the Plate Design window click Source Plate in the protocol tree Click Edit to enter the template designer Plate Design Options Name 12 point curve Description 7 18 e eo 12 13 14 15 16 f1 718 1920 Uses 5 mM source concentration with final assay volume set to 20 uL This example incorporates the controls and DMSO backfill in the destination plate Plate templates in this protocol Protocol El Source E Intermediate In
27. Compound 1C 250 250 2 346 36 379 65 A20 5 00E 07 M Compound 1C 2 5 2 5 2 346 36 377 65 Figure 3 42 Transfer report in csv format This report uses the default format however you can change the layout in the Options window Echo Dose Response User Manual 49 4 Software Reference This chapter contains descriptions of all the Echo Dose Response software screens This information and procedures for each screen are available in the Echo Dose Response online Help 4 1 4 2 4 3 4 4 45 4 6 TOO Do oa o 51 ID arene te ee ee E ee ee 52 4 2 1 Source Plate Designer oooooncococnoccconooncconoonnoononnconnonononnonn noo nonn non nconnonnons 54 4 2 2 Intermediate Plate Deere 58 4 2 3 Destination Plate Designer cccccscscsscsscssscsssssscesssscsessessesteesseeseees 62 PPOO CORON a 65 Al RUSS area 66 oe ae CC i 70 elec ete A 13 Dale e Oe 11S aaor eunmscanontacneachaseeateaes 73 AAZ Pate 1 2 201 0 ee ee 75 AAS E A 78 LA tment te te ECO E 79 Bat e PE o AAA 80 E Al A PU 82 ADS RIGO eonenna anaa 82 A a e Si A A A AA 87 Echo Dose Response User Manual 50 4 1 Toolbar The Toolbar contains the File Tools and Help menus as well as individual icons for frequently used functions Labcyte Echo Dose Response File Protocol Tools Help De a o gt From the oo bar you can perform the following functions File menu File Protocol Tools Help Connect to Instrument Connect to instrument S
28. Design the Source Plate Template Defines the source plate type and assigns wells to compound control or DMSO See Step 1 Fields and Buttons below Step 1 Design the source plate template Select the wells in the microplate and then click on one of the buttons in the Legend to define the type for the selected wells in the source plate template When finished click on Next Q TEE EEIEIE EIEEEI EEE 54 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 000000000000000 Figure 4 3 Source Plate Designer Step 1 Fields and Buttons 2 x 000000000000000 000000000000000 cd Source Plate Type Drop down menu of available source plates Source plate must be an Echo qualified plate Plate map of the selected source plate well positions that can be clicked and assigned to the components in the We Selection Action box Well Selection Action Components that can be assigned to the selected well positions Assigns one or more compounds or compound concentration Opens a prompt box to enter the compound concentration Compound Conc El Control Assigns negative positive or other control compound DIMSO Assigns wells to hold DMSO for backfilling assay wells in the destination plate TERN Prevents liquid transfer to or from the selected wells Reserved C EuR By default all we
29. E 03 M 25 0 nL 504 uL 4 995E 06 M Figure 3 15 Intermediate concentration Note The range of the new concentrations you create depends on the working range of the source plate you use The plate with the widest range is Labcyte 384PP StdV which has a working range from 20 uL to 50 pL For working ranges on other source plates go to www Labcyte com click the Products menu select Consumables and open the Web page for Echo qualitied microplates 8 Click the Next button and enter the intermediate template name Intermediate Dilutions Plate 9 Click the Finish button to return to the Pate Design window The software will create additional source concentrations for each compound The following figure shows 32 wells assigned to the intermediate compound concentration two concentrations for each compound and several wells assigned to hold bulk DMSO DMSO 1 HOCOSSSOOSSOCOOO Neeeeeeeeeeeeeoee ay dod dd dot dad USO e HOSS CCCO 00000000 ESOO OOOO OOOO OOOO O OOOO HOCeeeeeeeeeeeeee OOOO OO HOOD OOO DO OOOO OOOO O0OOOOO OOOO H eeeeeeeeeeeeeee See OOO Heeeeeeeeeeeeeeee HOSS eeeeeeeeeeeee HOOO OOO OOOO H CCeeeeeeeeeeeee HOS CCeeeeeeeeeee OOOO 490 000 000 000000S DSSSODOSAOOSSSaSo HS DOS SOLO lt lle lo fo fu fu el 1 gt lt l lo 8 1 1 1 Compound 1 Compound 2 Compound 3 Figure 3 16 Intermediate plate design The intermediate template is done 29 Echo Dose Respo
30. LABCYTES Dose Response Software Lat ation Plate Litera Step 2 Dele the dose response data points Select ihe number of dala ponia and the number of combi por dosd 21ponid Curd Please fil oui the table below in its entinety Fal Dose Pissporss Lunes Cathet 2438505 M 7 aE i Feide Combet r Dropdcan Lahi e 2112504 4 ve Sain o Vis aa pl eae 1 0 User Manual LEDIN home DMSO Baciha e Nurser of dati contre parir cer curve Le 7 AEDEM Pelo Becki O fmi Lo nson Software version 1 2 7 SEs M E cacao o O Sune Puto 7 450 10 M O Diet Fates Lec 0005 00 M cure Mi Decio corpaurd Al Deacon A O AS A AA Daibit h Flair Diriats Step 3 Choose a name dos ite destination plate temgplala Tha meme o thes templada muni be regu within they prem Ticos hiie Peel Sere lion A 0 EET 095 0 OO oe ao CE OE OOE E de LEE E E EEE E EE E E E E dos eB e bed tee bb Pete bb hee hea he iia y E Ca E E ee ee iais is a aaa s ais a aaa aal i Saa ais A A a ae a a a E E E E te ped a E E E E E a a hae eas tE Ethet htta eeth ee alasali a a alas ali aa aa a a dra e e a RARE Pe ee a ae PREP REPRE PRED Ep Pee pe eh HEPES PEPE EEE EEE EE Pe AAA ee E E E E E E E E E E E E E E E EET eh dd ducted dake AAA ee ee Oke Pde i dee Ae aks Pd ed Ad AAA ee ee A HH Hp eee ie rl Ari di 2 Fe LALA i ai p d po
31. Map 1 eegee 0900000009 00000 COSO 000000000020 0CUDODS ransfer from Source Plate 1 46400 0880080888000 00500 ee ee ae LISS SSO 6 OSSOSSOS8O8O 0900008 Transfer to Destination Plate 04 7 e bh eee ese te ee Qo etc E Compound G6SOSO80SS888E8O09 259 C9000 Prefetch Plates Sees 99OOOS09998209099090 EJ Control Do hon Wake a C omso 7 Destination Piate 04 Grener_984PS_781096 1 Source Plate Labcyte_384PP_StdV 384 Spots Destination Plate Type E Greiner_384PS_781096 6 A SS II IES IIIT ICS ID ICI IC I EEE BOOS 2 Y ODOC OOO Seacceceenne e OOOO Y ODO Y OCOD ODPDOCOOD Soo al A a 209990 T Ssssss Frotocol CALabcyte Echo Protocols 12 pt w mult src edr rt ttt 2989298 E Source Plates 1 SES nados Intermediate Plates 2 Destination Plates 11 A Sessions 13 Figure 4 33 Protocol simulation with multiple source plates Once the simulation begins the software shows the number of intermediate and destination plates that are needed for this run upper right corner of the screen To process 4 source plates only 5 intermediate plates are needed If you were to run the protocol 4 times with one source plate per run you would require 8 intermediate plates See the following data comparison 98 No of runs 1 run 4 runs 1 run using multiple source plates Source Plates 1 4 4 Intermediate Plates 2 8 5 Destination Plates 11 44 44 Sessions 13 52 49 Figure 4 34 Plate
32. OOO 10000000000000000000000 00000000 OOOO PERES na o E ax aeeee0 eee E DMSO hes a aal 100000000004 vee a oo HNeeeeoeeeooose 900000000094 Beeeeeeeeoee NOCOSeeeeoees Figure 3 14 DMSO wells in source plate Echo Dose Response User Manual 27 Software Operation 28 Note The selected wells will be used as reservoirs for DMSO only During plate preparation or during the run when the bulk fill screen is displayed you will fill the intermediate plate with DMSO either manually or with a bulk filler The Echo system will use the DMSO in these wells to backtill the compound wells in the assay plate 6 Click Next The next screen provides fields to calculate the intermediate compound concentrations 7 Enter the following information Enter the number of intermediate concentrations you want to create In this example enter 1 A single concentration field will be displayed See Figure 3 15 Select the compound concentration from the Source Concentration menu Enter the transfer volume of the compound In this example enter 25 nL Enter the volume of DMSO you want to bulk fill 25 pL The software will calculate the final concentration of the compound in the intermediate plate Internediate transters How many intermediate concentrations would you like 1 i E al 1 3 Z Source Concentration Transfer Volume Bulk Fill Volume New Concentration 5 000
33. Plate Insert SOURCE PLATE W 2 10 MM INSET Barcode e Verify Barcode Echo Dose Response User Manual 39 Software Operation 3 4 Run a Simulation What is a simulation The simulation program is a computer generated execution of the transfer protocol When you click Simulate in the Aun Options window the software displays a mock run window that shows the source and destination plate maps protocol information and run status and animation of the transfer Source or intermediate Destination Protocol data Transfer plate map 40 Compound 1 and run status animation Screening in 1536 well plate edr Simulator C L 6 well plate edr 4 b gt Source Plate Type Protocol ALabcyte Echo Protocols Screening in 1536 well Labcyte_284PP_StaV Source Plates E Oe FELEPITETT Destination Plate Type Greiner_ 1536HiBasePS_ 782096 a ae eee et OOO E 1 13 ERRE EE dOOCODODOO Destination Plates 1 Sessions 1 Current Session 1 Current Transfer Map 0 Transfer from Compound and control Plate 1 Transferto 1536 well desination plate 01 2 fell Color Legend Compound GHSSGHSSSOSOGER CE Jeje el a 00060066606660 CTE Ier Io Ier o er o e 5 D D D D D ey D D D D PrefetchQueue b Se a Control ID Name Type Wells Barcode DMSO 2 1536 well desination plate 01 Greiner_1536HiBasePS_ 782096 1536 1 Compound and control Plate Labcyte_384PP_
34. Plate in the protocol tree Click Edit to enter the template designer Plate Design Options Name 12 point curve Description Uses 5 mM source concentration with final assay volume set to 20uL This example incorporates the controls and DMSO backfill in the destination plate 7 Js halisfieli7 ialis Plate templates in this protocol 5 O 12 ER oe 009 A OOOOOOO OO OODO O O W OAO 9990000999990 e 15 Secnees OO0OOOO canoas 0000000 00000000000 rr oror o 8 Protocol Plate Template Actions Source Source Plate Add Source Intermediate PS Destination 3 E a See e gt gt a e E gt e a0 ee e Destination Plate Pole REE Elo IristoleP M OO DB OOOOO OGM OO B OOOOO M DO DODDOOQGBDOGOOOO_ RABIA B O DOOO B OOOOOOOD B O DOOO OBOOOOGOOD Edit Figure A 9 Preview of intermediate plate template 5 Create the destination plate template a Click the Add Destination button in the P ate Design window to open the Destination Plate Designer window b Select the plate type of the destination plate The controls will not be entered at this step because they will be included in the dose response curve c Click Next to continue to Step 2 This screen calculates the dose response curve The data you enter will define the layout of the final assay plate d Ente
35. Response directory You will need Adobe Acrobat Reader to open this document H m Labcyte j pr Echo Dos hin Ciara Response ES El Echo Dose a tal All Programs Quick Start start O O 3 Figure 2 8 Location of Quick Start Guide IC50 Calculator The Echo Dose Response Calculator is a soreadsheet that contains sample IC50 curve calculations It includes interactive fields to help you determine volume requirements for compound and DMSO What is the concentration of your source solution What is the final volume of your assay can tell you how to set up the following IC analyses for you with your Dose Response software 12 Point 5 48 Log 6 Point 5 Log low DMSO concentration 9 Point 4 82 Log halving concentrations low DMSO concentration 19 Point 5 42 Log uses more sample can set up a 12 point six log ICs analysis for compounds of your choice at these concentrations Highest concentration Lowest concentration 9 10 11 12 75 The highest concentration of DMSO is 0 38 Is this DMSO concentration too high You can reduce the concentration of DMSO by increasing yd Total sample used 75 25 7 5 2 5 250 2 5 362 5 nL Sample used for triplicate analysis 3X 75 25 7 5 2 5 250 2 5 582 5 nL Total DMSO used with backfill 25000 25000 3X50 3X67 5 3X72 5 50 57 uL 51 71 if triplicate 1 10 000 Stoc 2 5 nL 25 uL Total DMSO used no backfill 50 pL 7 8 Figure 2 9 1
36. StdV 384 Empty GOLOGOGIROOANOL 890000000000000L See eee E DOADEOO BOOVO DO jew Full Size Zoom In View region of plate 2 aj 2 Transfer from P2 to A18 Skip Animation Play Close Figure 3 29 Run simulation screen Q QULVCOSSSOOOOOGOLE QUVOOOVTGGSSOOGOE SIGOOOOGLCOCOSBOE BOSHOOGGOOOOOOOE BOLVOSSSBOOOOSOOE BOBOOOOLOOOO The simulation is very useful for testing a newly created protocol and should be run before committing time and material to the actual transter run Because the simulation runs very quickly you can test several different plate configurations The simulation can also be used to assemble the plates needed for the transfer protocol The protocol data in the upper right area of the screen displays the number of source intermediate if used and destination plates that are needed to run the protocol For an in depth discussion on how the simulation works go to page 70 To run a protocol simulation 1 Click the Run icon or select Run from the Protocol Protoco menu If you have not saved your Error Check protocol you will be prompted to save it now cule S 2 Enter the Number Of Full Source Plates that will be used in this run 3 Click the Simulate button in the Aun Options window Run Options ECHO 1 gt ES55x40791 Available Normal gt Full plates as defined in protocol Number of Full Source Plates 1 Partial plate
37. Transfer Protocols Preface Manual Conventions Some of the text in this manual uses special formatting to indicate emphasis or keystrokes The text conventions are described below Indicates an on screen button Bold text Press the Next button label menu title or menu item Courier client txt Indicates a display output Clvent 61d2Z txt printed output keyboard input or file name Blue underlined Software installation Signities a hyperlink to a topic Quotation marks and See Set up Preferences Refers you to a topic elsewhere italics on page 14 in the manual Usually includes a hyperlink vi 1 Introduction The Labcyte Echo Dose Response software automates the tedious process of preparing active compound concentrations for IC50 Dose Response analyses This software works seamlessly with the Echo Liquid Handler to pick up nanoliter quantities of compound from the source plate and then transfer them to the assay plates Laboratory time and expense are reduced through the following key features e Direct transfer from the library plate to assay plate in concentrations required for the dose response analyses e Automation of the transfer process thus increasing accuracy and eliminating cumulated error during serial dilutions Significant reduction in compound and DMSO volume by using microplates instead of test tubes Dilution can be performed in 96 384 and even 1536 well plates e Cost savings
38. UD DEVELO CS rated 14 RI 16 Software Opera air 17 o etree 18 32 Delo ihe Pre NSIC PNG OCO etna PO 0U 0C OP PPP PP 19 oli A e PP ee ee 20 3 2 2 Create me source plate template escencial 21 3 2 3 Create intermediate plate teMplate coonicnncnnnccconooccnononnnnononnnnnnons 27 3 2 4 Create destination plate template iene 30 3 2 5 Specify order of transferring COMPOUNO cccocccccncononnninnnnnooncnnconnonnnononanns 36 O ASS DOTO CO ere O T 37 E E OE EE O een ee ere 37 A 38 3 4 Run a Simulation ooccccnnoconoccononononnnonnonnnonnonncoononnnon nono non nor non nonn non nnnn nana cnnnnnnns 40 SHOES UI eee 43 E a Ue o CO a ahi OO oa 44 3 7 Manage Transfer Data ccccccccccsscsccsscsscssessecsececsesssccsecsessesseseessesaessessaseaeeas 48 SoftWare Referte aia 50 A NE 6 0 o Ea 51 a i aD A PA e O ee ee eee 52 4 2 1 Source Plate DesigN8T oonoccoconnococonoonccononnnononnnoononn noo nononoononnnonnonnnonnons 54 4 2 2 Intermediate Plate DesigNeT o oocconccconnoccnonoocnnononnnnnononoononnnconononcnnono 58 4 2 3 Destination Plate D SIQnel cccccccscscsecsecsecsesssscsesseecesseessesteesseeeees 62 O 10 GO Ul weeedcese a yesiu DEI oes 65 l RUN o O A 66 NS A 0 OO OE en ee eee ee 70 E E OE EO EEE 73 Ales on Mn 73 442 Plate Type Ed 75 A ee 78 O O 79 AP e 80 AA O DO all Oe op o E E ee eee eee 82 LO A o ee ee 82 A Pee ee nee o on A 87 APPEAR 88 SEU A 88 Example 2 12 pt curve using multiple source plates
39. acent to the original curve Curve Fill Direction Transfers concentrations of the same compound across or down the plate 10 curve concentrations for compound 1 Figure 4 11 Curve fill across Compound Fill Direction Transfers different compounds across or down the plate Compound 1 Compound 2 A e lll dads Le 7 Pe 9 hobufbahsthsis17 18 1920821 feels fe Figure 4 12 Compound fill across Step 3 Choose a name for the destination plate template window Provides a name for the Destination Plate and displays the final layout Destination Plate Designer 21x Step 3 Choose a name for the destination plate template The name of the template must be unique within the system Template Name Destination Plate Plate Type Labcyte_384PP_StdV Plate Preview 1 23 4 ls e 7 e T hofaJ2 As rs 17 18 191B0 12112212324 BORE ERE REORDER CE Figure 4 13 Destination Plate Designer Step 3 4 3 Protocol Run The Aun Options window enables you to run a simulation of the transfer protocol or run the transfer protocol on the Echo liquid handler If you are not connected to an instrument this window also provides a connection dialog box axi Instrument EcHo1 Connect Disconnect Serial Number E55x 0791 Status Available Normal Protocol Name CALlabcytetEcho Protocols 10pt_assay_ with_backdill_w12 edr Run Protocol Full plates as defined in protocol Number of Full S
40. backfill in the destination plate 7 ILe Js Jhro 145 s t7 h8 19 Plate templates in this protocol Protocol m Plate Template Actions Source 3 Source Plate d Source Intermediate AO Intermediate Dilutions Plate Destination Destination Plate OOOO Cz OOO OOO OOOO OL OOOO OL QOOOOOOOOOOOOOOOo na 3 SO 3 3 3 re 3 3 s s 20 oo DO 00 o0 oe BE OPGCOCOOC0QOCOOCOGOL OPGCOCQOCOCOGOL OPGCOCQOOCONOL QQBOQOEW OO OOGO SOSOGSOVOSGOGOGAL dOSOGSOVOGOGVOGAL OOOO a 14 15 20 35 aeee O Q Q 133 ae 3133 208808 9 Q Q E O Q 882202202 OPGCOCQOOCONOL POGQPODOEWDOEO E QOEOO eee 000800808080808880O00000 a a a a M a 00 ae OO OO QQ QQQ QQ OO Edit Figure 4 31 Preview of destination plate template 6 Inthe Pate Design window specify the order of processing the plates Automatic The software selects the most efficient order of plate handling This is the default setting and may be fine for most protocols Source plate centric The software minimizes the frequency of swapping the source plate This option is useful if you need to complete the processing of the source plate and return it to storage Destination plate centric The software minimizes the frequency of changing the destination plate This option is best if you
41. bles you to limit the volume ot DMSO if your assay cannot handle too much DMSO The software calculates the backfill volume of DMSO and the final compound concentration for each data point and then adds them to the dose response data point table Enter the maximum volume of DMSO to be filled by the Echo liquid handler 60 nL Fill volumes greater than 60 nL must be backfilled with a bulk filler Dose Response Curve Number of data points per curve 12 Include Controls in Dropdown Lists E Assay Volume added to each well 25 00 pL Automatic DMSO Backfill V Perform Backfill C First Last MW Maximum Backfill Volume 75 0 nL M Backfill offline for volumes greater than equal to Yellow denotes offline backfill soo Figure 3 20 Dose Response Curve box The backfill offline volume gives you control over how much DMSO to fill with a bulk filler The bulk filler can dispense larger volumes more quickly than the Echo liquid handler Backfill volumes greater than or equal to the backfill offline volume 60 nL in this example are highlighted in yellow in the data points table Please fill out the table below in its entirety Source anser Backfill Volume a Concentration Drees inl olume oOmpoun ul Figure 3 21 Data point table showing offline volumes Note The accuracy of offline back tilling depends on your bulk filler If you need more accuracy than your bulk filler can provide for example 92 5
42. d e f you purchased the Echo Dose Response software separately then you will have to perform this task To activate the instrument 1 Start the Echo Dose Response software 2 Inthe Connect to Instrumentscreen select an instrument that you will be using with the Echo Dose Response software and click the Connect button Connect to Instrument Type or select the name of an Echo instrument and click on Connect Work Offline Connect Cancel Figure 2 11 Connect to instrument 3 Goto the 7 00 s menu and select the Activate Tools Help Instrument option Preferences Labware Definitions Actvate Instrument 12 4 Enter the license key The license key is an encrypted block of text You can insert the license key into the Activate nstrument window in one of the following ways Copy the license key from the file you received from Labcyte and paste it into the License Key text box Click the Open License Key File button and browse for the license key file Activate Instrument y xj Please type the name or IP address of the Echo instrument that you wish to activate and either copy paste the license key in the box below or click on Open File to select the file containing the license key Instrument 7 Se al Humber ES5 0691 Status Available Normal Disconnect Get Current Key license Key EEY START oUS5vHyRADbCeHJdAgT3Bduqvi LIOtm HO tm rceCUB3Npd
43. destination plate swapping Figure 3 27 Process plate options Hint You can save processing time by turning on the Survey History function This function saves survey data for the source plate and reuses it the next time that the source plate is returned to the Echo liquid handler For more information go to Survey History page 80 For this sample protocol use the Automatic option Protocol Tools Help 3 2 6 Save the protocol Connect to Instrument 1 Open the F e menu in the 7oolbar New cana Gr Open Ctrl 0 2 Select Save 3 Enter a file name in the prompt box The protocol is saved with the file extension edr in C Labcyte Echo Protocols the default directory You can save the file to a different directory 4 When you edit the template an asterisk appears next to Dp eulelb gt the file name This is a reminder that the file has information that has not been saved E 3 2 7 Check for Errors The Echo Dose Response software contains many checks and prompts during the creation of each plate template However it is useful to do a check of the entire protocol before running it This program looks for inconsistent information e Select Error Check from the Protoco menu Protocol Tools Error Check The transfer protocol is done Run Echo Dose Response User Manual 37 Software Operation 38 3 3 Select a Protocol Run The Aun Options window opens when you click t
44. detailed description see Protoco Options on page 79 Workflow and template preview The worktlow diagram is displayed whenever a protocol is open It serves as a reference tool for planning the protocol When you select any template in the template tree left pane the plate design for the template is displayed in the right pane Status bar The status bar displays Echo connection information such as instrument name and serial number and the version of Echo client software that is running Navigation and Exit buttons The left and right arrow heads 4 t become active when you have too many protocols open to view all of the file names The arrows shift the protocol tabs left or right to display the file names The Exit icon x closes the displayed protocol Echo Dose Response User Manual 11 Getting Started 2 4 Software Setup Your Echo Dose Response software needs very little setup It comes with default settings such as output file type and report format that can be used If you want to change these settings refer to the tasks listed below otherwise continue to the next section Software Overview on page 17 2 4 1 Activate Instrument To run the Echo Dose Response software on an Echo liquid handler you will need to obtain a software license key from Labcyte e f you purchased the Echo liquid handler and Echo Dose Response software at the same time your instrument will be delivered with the key pre installe
45. e using the same plate ID for two different plates and display an error message Note If you are running a pick list and you selected Pre process pick list in the order specified in the Aun Options window the software displays the source plate barcode or ID that should be inserted during the run In this situation the Barcode field cannot be edited You can disable barcode verification by deselecting the Verify Barcode option after you have entered a barcode or plate ID in the Barcode field You can also skip the Barcode field entirely and click OK This sample protocol uses only one source intermediate and destination plate therefore you will skip the Barcode field Follow the directions in each plate prompt and click OK The Aun Status window will continuously update as the transfer process continues When the run reaches the bulk fill step the software will display the Bu k Fill prompt box BulkFill Intermediate Dilutions Plate with DM50 01 gt Name Intermediate Dilutions Plate with DMS0 01 Ls Type Labcyte_384PP_StdW Wells 384 Fill Column Column Volume Units 1 30 uL 2 30 ul 3 30 uL 4 30 uL 19 30 uL 20 30 uL 21 30 uL 22 30 uL 23 30 uL 24 30 UL Ok Cancel Save As Figure 3 38 Bulk fill status during transfer run You can use this information to pipet DMSO into the bulk fill wells of the intermediate plate If you prefer to save it to file for example to print it use the
46. ect the number of data points and the number of controls per dose response curve Please fill out the table below in its entirety z Transfer Vol Dose Response Curve Dai E a hb Assay Volume uL Final Concentration entrat Number of data points per curve 5 5 000E 03 M 250 0 5 000E 05 M T 5 000E 03 M 75 0 1 500E 05 M Assay Volume 25 00 uL 5 000E 03 M 25 0 5 000E 06 M 5 000E 03 M 75 1 500E 06 M 5 000E 03 M 25 5 000E 07 M Automatic DMSO Backfill I Replicates Same Plate Number of Replicates 2 C Different Plates e Across Across C Down C Down E Fill Direction m Compound Fill Direction gt Cancel lt lt lt Previous Figure 4 10 Destination Plate Designer Step 2 Echo Dose Response User Manual 63 Software Reference 64 Step 2 Fields Data Point Table Table of compound concentrations transfer volume assay volume and calculation of final concentration Dose response curve Data fields to specify the number of data points in the dose response curve assay volume needed for each well and option to automatically backfill the wells to the assay volume Replicates Data fields to specify the number of curve replicates on the same plate or across separate plates single plate per curve and its replicates In the same plate option replicates can be added after the last curve in the plate or placed adj
47. elect an Echo liquid handler to connect to the software d nd New Create a new transfer protocol y eu ue Open Open an existing transfer protocol Save colts Save Saves the current transfer protocol Save As Save As Copies the current transfer Exit protocol to a different file name Exit Closes the Echo Dose Response software Protocol menu Protocol Tools Help Error Check Checks the protocol for inconsistent Error Check information Run Crl k Run Executes the transfer protocol that is displayed Tools menu Tools Help Preferences Set survey output and report Preferences options for all protocols Labware definitions Manage existing Ale LE labware definitions or add new definitions Activate the instrument Enter software license key to use the Echo Dose Response software on each Echo liquid handler Actvate Instrument Available only when a protocol is open Echo Dose Response User Manual 51 Software Reference Help menu Contents View the Help table of contents Contents Index Search Help through available key words Index Search Search Help through words or phrases oe About Display the version number of the Echo Dose Response software About 4 2 Plate Design The Plate Design window contains the protocol name description and plate templates Plate Design Options Name Screening in 1536 well plate Description 384 source plate conta
48. ermediate Dilutions Plate with DMSO Destination Destination Plate Figure 4 2 Plate Template Tree Plate Template Actions Add Source Opens the Source Plate Designer wizard to create the source plate template and add it to the Protocol tree Add Intermediate Opens the Intermediate Plate Designer wizard to create the source plate template and add it to the protocol tree Add Destination Opens the Destination Plate Designer wizard to create the source plate template and add it to the protocol tree Edit Opens the selected plate template in the protocol tree for editing Delete Deletes the selected plate template in the protocol tree Plate processing order Automatic most efficient order of compound transfer Default setting Source plate centric minimizes frequency of changing source plate This option is useful if you need to complete the processing of the source plate and return it to storage Destination plate centric minimizes frequency of changing destination plate This option is best if you need to complete the processing of the destination plate and put it in environmentally controlled plate storage Echo Dose Response User Manual 53 Software Reference 4 2 1 Source Plate Designer The Add Source button begins the Source Plate Designer wizard in a new window This wizard helps you prepare a template for transferring compound trom the source plate to one or more destination plates Step 1
49. erred to the destination plate Figure 3 24 shows the location of each compound and its dilutions for the Curve and Compound fill directions across down IC Curve fill Conc 1 and 2 Conc 3 from source plate from intermediate plate Compound fill C ECCELCE cra ofzf elr ale z e m o Lolo gt SAO Figure 3 24 Destination plate design Figure 3 25 shows additional configurations for curve and compound fill directions The option you choose may depend on your testing requirements 1 Curve Fill Across Compound Fill Across 2 Curve Fill Across Compound Fill Down I Te e ais Te I Te Leholsfefsprsfsfrefa7frsfiofeofzifeajesyas Eiee fe I Te gt hot 5H 17 relt of2o 212223 DODDODODDDDODD VPO WDODOODO 0000000 QQ D2 VDOOOVDO DOGO VDD OOOGOOD Bee esse sere 9opororor 00090000 1 I1 Cees 4 D O da 49 O O Q O 9 Q Q o Bibi 1600000000000 00060I000 DQOOQOOD Figure 3 25 Curve and compound fill options Note The software fits all compound concentrations for a curve on the same plate If a complete curve cannot fit on the plate it is moved to the next plate In configuration 3 the curve is filled down the 384 well plate There is insufficient vertical room to fit more curves therefore only 12 curves can fit on the plate for this protocol The other three configurations fit 16 curves Echo Dose Response User Manual 35 Software Operation 36 When you ha
50. fer to destination plates Flexible e Places control compounds in desired wells specified by the user Maps the design from any plate to any plate type e Provides replicates on the same or separate assay plates Allows user defined curve orientation Processes dose response curves from a hit list Allows the output files to be run specitic or destination plate specific Generates output files in XML CSV and TXT format Accurate Eliminates accumulated error from serial dilution Automates manual steps e Removes washing steps Maintains survey history Echo Dose Response User Manual 2 Getting Started This chapter describes the basic tasks to start set up and finally exit the software 2 1 Software Startup To start the Echo Dose Response software 1 Open the Start menu 2 Select All Programs or Programs gt Labcyte gt Echo Dose Response 3 Select the Echo Dose Response program Mina Y Administrative Tools ag Labcyte Calibration 4 Adobe Acrobat PEA 5 Internet Explor Microsoft Office Tools FA Microsoft Works k MIU OSUL ALLES All Programs Echo Dose Response Microsoft PowerPoint Figure 2 1 Location of Echo Dose Response Software Hint To create a shortcut to the Echo Dose Response software on your computer desktop press and hold the Control key while you drag the Echo Dose Response icon A to the desktop The software begins with the Connect to Instr
51. fore you start the Echo Dose Response program have the following information ready Initial concentrations of your compound Number of compounds Location of controls if they are used control samples can be on the compound source plate separate source plate or destination plate Layout for the source plate Layout for the intermediate plate if additional compound concentrations are needed Final assay volume Number of points needed for the IC50 curve You may already have a calculation utility to help you with the 1C50 curve If not try the Echo Dose Response calculator in the Echo Dose Response directory The calculator contains sample curve data that shows the volume of compound and DMSO needed for different IC50 analyses Also see the sample protocols included with your software for examples to follow For this sample protocol the following data will be used Starting concentration Original compound at 5 mM Layout for the compound source plate 16 compound wells two columns in 384 well plate Layout for the control source plate 32 control wells Layout for the intermediate plate two columns in 384 well plate Two dilutions of stock compound in 384 well plate 1 100 1 10 000 Number of points needed to plot the IC50 curve Assay reagents added to each well 25 uL 3 2 Set up the Transfer Protocol The Echo Dose Response software is very flexible It enables you to pick compounds from any well position in y
52. gress you will need to cancel it before you can close the window The Source Plate Type graphic upper left shows the source plate that is selected for transfer Source Plate Type Labcyte_384PP_StdW AE A e A 0 0 61 6 16s 0 76 6 JE JE JE SI L L L OA OIR AI A As the simulation runs each source well shows the number of times the compound is aspirated In this sample graphic the second source plate is in the process of being aspirated 5 times The destination wells in red were transferred trom the first source plate The Destination Plate Type graphic lower left shows each well that receives a dispense and the number of dispenses that are received In this graphic the destination plate receives a single dispense When the dispenses are complete the well color will turn red Destination Plate Type Labcyte_384PP_5tdW E li ls o ln ee EE CJE e e i DO i O O O i O O O l aF ak DA A GD A E GP GD A GG GP E O 4 A ul a Ge e GO 46 de 69 46 El 59 40 ER 59 40 GG GP 0 0D E Gs neet TTTTTT ih lt p a i b u ca za L Echo Dose Response User Manual 71 Software Reference 72 The Well Color Legend middle shows the colors that correspond to the compound being transterred red control cyan DMSO yellow and Empty wells that have not been aspirated or dispensed Dark blue wells represent wells that are in the middle of the transfer process When the transfer is complete the well changes to
53. h survey history can be used indefinitely it is not recommended Echo Dose Response User Manual 81 Software Reference 4 5 2 Output Files The Output Files box in the Options window organizes the survey and transfer results according to the following criteria Output Options Output Files f Run specific Source Plate specific affected by processing order Figure 4 23 Output Files options Run specific All protocol data is stored in one file in the Process plates in this protocol order that the results are generated Automatic Plate specific Protocol data is stored in separate Tiles Source plate centric based on source or destination plates depending on which Destination plate centric process order you specified in the Pate Design window 4 5 3 File Type Survey and transfer results can be saved to the following file formats that are compatible with most laboratory information systems XML extensible markup language CSV comma separated values TXT text file The results will be saved in the formats you have checked and stored in the directory you specify in the Directory option 82 Report Format The survey and transfer reports can be customized by clicking the Format button in the Protocol Options window Note The DEFAULT settings in the Transfer and Survey Reports are from the Preterences Options window Plate Design Options Protocol Options Surveys
54. he Run Ele Protocol Tools Help icon in the Echo Dose Response toolbar This window aja IES gives you two run functions Simulate and Run 3_compound_assay Run 2x Instrument echo1 Connect Disconnect Serial Number E55X 0791 Available Normal Protocol Name EC iLabcyte Echo Protocols 10pt_ y owith_backtill_v12 edr gt Full plates as defined in protocol Number of Full Source Plates 1 Partial plates as defined by a pick list Figure 3 28 Run Options window Simulate The S mu ate option opens a mock run window to allow you to simulate the protocol on the computer before performing the actual transfer This option is available when you are working offline or when connected to the Echo liquid handler Go to Run a Simulation for more intormation and procedures Run The Aun option opens the run status window to run the transfer protocol This option is available only when you are connected to the Echo liquid handler The prompt box contains a Connect button to communicate with the Echo liquid handler Go to Aun the Protoco on page 44 for more information and procedures The Aun Options window also contains the following options Connect Disconnect This option enables you to connect to the Echo liquid handler directly from the Aun Options window instead of opening the Connect window After you click Connect the software displays the instrument serial number and availability normal or bus
55. iate Dilutions Plate 01 19 Source Plate El 0 005 M Intermediate Dilutions Plate 01 20 Source Plate F1 0 005 M Intermediate Dilutions Plate 01 21 Source Plate F1 0005 M Tol 22 Source Plata 61 0 005 M Intermediate Dilutions Plate 01 23 Sy Plate ry T i a m Source Plate 4 Source Plate 6 Source Plate PT Source Plate Destination Current Current Destination Destination Concentration Compound Transfer Actual Transfer Fluid Fluid Well Concentration Units Name Volume Volume Status Height Volume DMSO Al 4 95E 05 Ml Compound 1 250 250 2 346 36 379 05 AZ 5 O0E 07 Ml Compound 1 2 5 2 5 2 546 36 377 05 AS 4 95E 05 M Compound 2 250 250 2 346 36 379 05 AA 5 00E 07 M Compound 2 2 5 2 5 2 546 36 377 05 AS 4 95E 05 Ml Compound 3 250 250 2 346 36 379 65 Ab 5 00 07 M Compound 3 25 2 5 2 346 36 377 65 AT 4 95E 05 Ml Compound 4 250 250 2 346 36 379 65 AD 5 00 07 M Compound 4 2 5 2 5 2 346 36 377 65 Ag 4 95E 05 M Compound 5 250 250 2 346 36 379 65 A10 5 00E 07 M Compound 5 25 2 5 2 346 36 377 65 A 1 4 95E 05 M Compound 6 250 250 2 346 36 379 65 A 2 5006 07 M Compound 6 25 25 2 346 36 377 65 A13 4 95E 05 M Compound 7 250 250 2 346 36 379 65 A14 5 00E 07 M Compound 7 2 5 2 5 2 346 36 377 65 A 5 4 95E 05 M Compound 6 250 250 2 346 36 379 65 A16 5006 07 M Compound 6 2 5 2 5 2 346 36 377 65 Al 4 95E 05 M Compound 9 250 250 2 346 36 379 65 A o 5 00E 07 M Compound 9 2 5 25 2 346 36 377 65 A19 4 95E 05 M
56. ins three concentrations for each compound 10 mM in quadrant Al 0 1 mM in quadrant AZ and 1 uM in quadrant B1 Two controls and DMSO wells are included in the source plate 1536 destination plate contains 13 point curve including controls Total assay volume Plate templates in this protocol Plate Template Actions Add Source Add Intermediate Protocol Source Compound and control Flate Destination 1536 well desination plate Add Destination There are 3 source concentrations per compound in this protocol Process plates in this protocol f Automatic Protocol defines the optimum sequence Source plate centric Minimize source plate swapping Destination plate centic Minimize destination plate swapping Figure 4 1 Plate Design Window 52 Name Name of the template for the transter protocol This is a user assigned name that is different than the filename Description Additional text that describes the template such as the list compounds that are being transterred concentrations number of points on the dose response curve and controls and backfill if they are used Plate templates in this protocol List of plate designs templates in a a rectory tree that are used in this protocol Double click the template in the directory tree to open it Plate templates in this protocol Protocol HO Source Source Plate Control Plate Intermediate i Int
57. ks wells from compound transfer See Step 1 Fields and Buttons below Intermediate Plate Designer l E 2 x Step 1 Select wells to reserve or bulk fill with DMSO only Click on either the column heading or the row heading to select the wells in the intermediate plate Click on Reserved to exclude those wells from automatic processing or on Bulk Fill DMSO to fill those wells with only DMSO as part of a bulk fill operation When finished click on Next Intermediate Plate Type Laboyte_384PP_SidV y 213 fa is fs 7 sls prob 23114195 16117f18 roJ0 121122 f23f24 Seer seers NOOSOOCOCOOCOCOOCOCOO MN _oxraonso_ E900000000000000000009 B DOOCOCOOCCOOCOCOCOOOCOOCO P O OOOO mise E EDOOONODOOONNNNOOOOOOO00O DOOSCOOCOOOOCOCOCOCOCCO HOSCCOCOCOCOOCCOCOCOCOCOCOCOOS OOO OOOO 1900000000000000000009 OSOS Q000 Bulk Fill Direction e Column wise C Row wise Figure 4 6 Intermediate Plate Designer 58 Step 1 Fields and Buttons Source Plate Type Drop down menu of available source plates Plate map of the selected intermediate plate well positions that can be clicked and assigned to the components in the Well Selection Action box Well Selection Action Components that can be assigned to the selected well positions see below Assigns wells to hold DMSO for bulk filling compound wells in the intermediate plate or assay wells in the destination plate Bulk Fill DMSO Prevents liquid
58. lid choices 2 41 mm 6 10 mm and 7 62 mm See Note 2 below The width of the well opening at the bottom not an SBS specified dimension Valid range 0 0 to 86 0 mm The overall capacity of the well in microliters Valid range greater than 0 0 uL Currently the Echo liquid handler does not use dimensions F flange height and H well capacity Any value entered for flange height or well capacity is ignored Echo Dose Response User Manual 17 Software Reference 4 4 3 Plate Export The Export Plates window displays the plates currently available in the Echo Dose Response software You can export one or more plates to a file elw file extension for use in another application C Greiner_384P5_ 781096 C Labcyte 15235C0C_LoBase_ Dest C Labeyte_384C0C LV Dest C Labcyte_384PP_StdW_Dest C Labeyte LOW 384 Dest C Aurora 3456COC 00017829 C Greiner_1536HiBasePS_ 82096 C Coming _ 1536C0C_HiBase Dest Figure 4 19 Export Plates Window 78 4 5 Protocol Options The Protocol Options window is accessible by clicking the Options Protocol 1 tab behind the P ate Design tab This window enables you to customize the survey output and reporting options for the Echo 00002222 Dose Response software These settings are automatically used Plate Design Plone for any protocol you create The Protocol Options window displays the following protocol and output options Plate Design ptians Protocol Optio
59. ll enter the compound name and starting number in the Reolicate Pattern function Well Selection Actions DMSO Reserved Available Replicate Pattem Click on the button below after you have selected the wells to copy as compounds Replicate Selected Pattem c Click OK to close the prompt box The assigned well changes to red and is labeled a The a signifies the first concentration of the compound Hint You can move the cursor over the assigned well to view the compound name and concentration d Repeat steps a to c for the next compound concentration 0 5 mM The second concentration of the same compound will be labeled b and so on 4 Replicate the pattern To replicate the compound concentrations across a specific portion of the source plate a Select the wells you do not want to use for compounds and click the Reserved button to block these wells Some of the reserved wells will be used for controls and DMSO later on Note To select columns click the first column heading and drag the pointer to the last column heading Step 1 Design the source plate template Select the wells in the microplate and then click on one of the buttons in the Legend to define the type for the selected wells in the source plate template When finished click on Next Source Plate Type Labcyte 384PP_StdV bd Well Selection Actions Compound Conc
60. ll positions are available Use this vailable e button to deselect well positions that have been previously selected Selects all wells in the plate template for quick well assignment Same action as clicking the corner cell above well A Unselects all wells in the plate template that were previously selected Replicate Pattern box Replicates a selected well or pattern of wells to all available wells in the source plate Replicate Pattem Select the wells to copy Enter the base compound name and starting number click the direction to copy the wells and then click the button below Base Mame Kinase Starting Number 1 e Across Down Rename Selection Figure 4 4 Replicate Pattern box Echo Dose Response User Manual 55 56 Software Reference Base Name Applies a base name to selected and replicated compound wells Also replaces an existing name if the Rename Selection function is used Starting Number Begins the numbering sequence After the selected wells are replicated and numbered this field displays the next available replicate number Replicate Selected Pattern Replicates and numbers selected wells pattern in the direction that is specitied across or down Applies base name and sequentially numbers all compound wells Undo Replicate Pattern Reverses the last replicate function Rename Selection Renames selected compound wells with the text in the Base Name field Also re
61. mplate editing 3 2 3 Create intermediate plate template Typical transfer volumes for an Echo are from 2 5 nL to250 nL which translates to 2 logs on an IC50 curve You can use the intermediate plate to create additional dilutions on an IC50 curve that can span more than five logs 1 Click the Add Intermediate button in the Plate Design window to start the Intermediate Plate Designer wizard 2 Select the source plate from the Source Plate Type menu For example select Labcyte_384PP_StdV Note Since the intermediate plate will be used as both a destination plate and source plate it must be a source plate type 3 Select Bulk Fill Direction Column wise Before assigning wells to DMSO specify the direction that the assay wells are to be bulk filled The fill direction is dependent on the capabilities of your bulk filler Bulk Fill Direction Column wise C Row wise 4 Select the columns of wells that will be reserved for DMSO 5 Click the Bulk Fill DMSO button and enter fill volume in the prompt box 2x Step 1 Select wells to reserve or bulk fill with DMSO only Click on either the column heading or the row heading to select the wells in the intermediate plate Click on Reserved to exclude those wells from automatic processing or on Bulk Fill DMSO to fill those wells with only DMSO as part of a bulk fill operation When finished click on Next Intermediate Plate Type Labcyte_384PP_StdV MORO aaa ce AJO O
62. n Select a plate definition and click the Delete button You can delete a destination plate definition but not a source plate definition Import a single plate definition or group of definitions trom a specific file location The file must have an elw extension to be imported Export a single plate definition or group of definitions Reload the plate types that have been edited or deleted since the plate type list was last saved Save any changes you make to the plate type list such as adding a new plate definition Close the Plate Type List window 4 4 2 Plate Type Editor The Plate Type Editorwindow is similar to the Plate Specification window in the Echo liquid handler software Name Barcode Location Manufacturer Lot Number Fat Number Rows Columns Al X Offset A Al Y Offset B X Center Spacing E Y Center Spacing D Plate Height E Flange Height F Well Width G Well Capacity H Figure 4 18 Plate Type Editor Source plate definitions Source plates require very exacting specifications and calibration to accurately transfer nanoliter volumes therefore they are defined specifically for the Echo liquid handlers and tested at Labcyte Echo qualified For this reason source plates cannot be defined by the user For existing source plates only the barcode location can be edited Contact Labcyte to add more source plates Destination plate definitions The Echo liquid handler
63. n 1536 wellplate edr Run Status C 6 well plate edr 4 px Protocol Name C Documents and Settinos imiyasaki Wy Documents Echo 1C50 Screenina in 1536 well plate edr SOURCE PLATES DESTINATION PLATES Legend System Status Power Air Pressure a Tal ene Waming a Vacuum Pressure Y In Process Faut Coupling Fluid Y 9 saved EMO Waste Bottle Fluid Leak a Focus Calibration a 0 Pending Coupling Fluid Temperature 2 a RF Temperature 435 C m Transfer Status BER A Complete 50 Bapsed Time 00 01 29 01 Nov 2006 as 46 48PM Please insert Compound and control Plate Labcyte_384PP_StdV 01 Nov 2006 02 46 59PM Surveying plate 01 Nov 2006 02 47 00PM Please insert 1536 well desination plate 01 Figure 4 15 Run Status Window Control buttons Provides manual controls of the Echo liquid handler from the Echo Dose Response software Control Buttons 3 ps Opens the process door of the connected Echo liquid handler a Extends the source plate gripper Plate Out Destination BB Piate Out Extend the destination plate gripper Stops the transfer protocol that is in progress 66 Control Buttons HH Pause Pauses the transfer protocol that is in progress e Continue Continues the transfer protocol that is in progress Begins the transfer protocol Closes the Run Status window Source and Destination Plate windows Show
64. n Protocol Full plates as defined in protocol Number of Full Source Plates 1 Partial plates as defined by a pick list Figure 3 35 Run Options window connected to instrument 3 Click Run in the Aun Options window to open the Aun Status window Note If the Run button is inactive grayed out you are working offline Select an instrument and click Connect The Aun Status window is a dynamic window that provides control buttons to run the protocol It also contains various status windows to show the progress of the compound transfer the state of the Echo liquid handler and the final well positions and volume transferred Figure 3 36 shows a run in progress 16 compound assay edr Run St 16 compound assay edr Protocol Name C Labcyte Echo Protocols 16 compound assay edr SOURCE PLATES DESTINATION PLATES gt qe gt E Legend System Status O Intermediate e D1 oO 1 Power a Air Pressure a e Waming Vacuum Pressure JD In Process Faut Coupling Fluid EMO Y Waste Bottle Fluid Leak Focus Calibration a 0 Pending Coupling Fluid Temperature 22 E Emor RF Temperature 435 T Transfer Status ERDARA TRANSFER MAP o S Bapsed Time 00 00 36 Status Running 06 Jan 2007 04 42 43PM Surveying plate Labcyte_384PP_StdV 06 Jan 2007 04 42 47PM Survey Finished 06 Jan 2007 04 42 47PM Transfering 06 Jan 2007 04
65. nL then turn off the offline backfill option and the Echo liquid handler will perform the backfill for these wells 32 7 Enter the following information for this protocol in the Aep icates box Enter the Number of Replicates 3 Select the Same Plate and Adjacent Wells options Select the Curve Fill Direction Across Select the Compound Fill Direction Down Replicates Number of Replicates 3 Replicate Location Same Plate Different Plates Curve Fill Direction Across Down Compound Fill Direction Across f Down Figure 3 22 Replicate information In the Replicates box use the Same Plate option when you want replicate curves on the same plate use In Adjacent Wells option if you want the replicates to follow each curve Adjacent Wells option OFF TA Wells a ON GREE 00 007 SOVIVAIVIDA GAIL 20000000000000 F O L fleeces 3 2000003300 999999999 Da 100008 Ke Figure 3 23 Adjacent wells option Note The software fits the entire curve and its replicates onto the same plate If a curve and its replicates cannot fit on the plate they are moved to the next plate Use the Different Plates option when you want the replicates on different plates Echo Dose Response User Manual 33 Software Operation 34 The Curve Filland Compound Fil options provide flexibility in specifying the order that the compounds compound concentrations and controls are transf
66. name Echo Dose Response User Manual 85 Software Reference Filename Builder Form Figure 4 28 Survey Report Filename You can specify a different transfer or survey filename by clicking the Filename button in the Protocol Options window In Filename Builder Form window select a variable and click gt gt gt button to build the file name 86 4 6 Preferences The Preferences Ootions window is accessible from the 700 s menu This window is very similar to the Protocol Options window but with the following differences Labware Definitions Activate Instrument e Accessible trom the Toolbar When you click Preferences the Options window opens to display the following protocol and output options e Settings automatically apply to all procedures unless you make changes in the Protoco Options window report formats shown as default Labcyte Echo Dose Response Defaults 2 x Options Protocol Options Surveys Re Survey every source plate Maintain survey history Output Options Output Files f Run specific O Plate specific affected by processing order Transter Reports Save transter reports as file type lf xML M csv TXT Format Header 6 Footer 4 Directory C Labeyte Echo Reparts NameS SRunDates SinstrSN SRun Type ession Survey Reports Save survey reports as file type lf xml M csv TXT Format Header 6 Footer 4 Directo
67. nd function keys To create the source plate template 1 Click the Add Source button in the Plate Template Actions box to start the Source Plate Designer wizard The Source Plate Designer wizard is a series of software screens with instructions that take you through each step needed to create the source plate template 2 Select the source plate from the Source Plate Type menu For example select Labcyte_384PP_StdV See Figure 3 4 Source Plate Designer i q l Step 1 Design the source plate template Select the wells in the microplate and then click on one of the buttons in the Legend to define the type for the selected wells in the source plate template When finished click on Next jo TES Well Selection Actions AAA J Contral Figure 3 4 Source Plate Designer window step 1 a ee A A A ee A A A ee ee A ee A A a O Note The source plate must be an Echo qualified plate Echo Dose Response User Manual 21 Software Operation Selected e AI seececeeeooeeeeeee OOO well 22 3 Assign compounds to the source wells a Select the well that will contain the first compound The selected well is displayed with diagonal lines See Figure 3 5 b Click the Compound Conc button and enter the compound concentration and compound name in the prompt box In this example enter 5 mM and Compound 1 See Figure 3 5 Note If you plan to replicate and number this compound do not enter a compound name You wi
68. ng concentrations are needed to reach the full range of the curve See Figure 1 3 000000000000 IBIS 00600070000 anster 75 25 0000000000 and 25 d0dd0 4dd dd000 t four wells BOO8OOOOOOOO OODOCO00OODO0O OOCOOOOO0DODO Source Plate Assay Plate Transfer 75 25 7 5 Transfer 250 and 2 5 nL of each And 2 5 nL to diluted sample to two wells four wells OQQQQQQQQQQOQ Diute intermediate OOOO ONO Pie OOOO plate with 25 aL in 535660000000 Reece eee HERES peseseses Sky ES QOQOQ000000000 OQOOOOOOO000O OO0ODO00DODOJDOD O OOCOOOOO0OOD00O OOOOOO00O000O Intermediate Plate Figure 1 3 Direct compound transfer for dose response curves In the example above the Echo system is used to create an intermediate plate with two additional starting concentrations The system then transfers compounds from the source plate and intermediate plate to the assay plate The assay plate now has sufficient compound concentrations to create a 12 pt dose response curve All of the steps in this example can be accomplished by the Echo system using a single Echo dose response protocol with a high degree of accuracy and precision User intervention is held to a minimum 1 4 What are the benefits The Echo Dose Response software provides the following benefits Fast e Sets up more than 400 12 point dose response curves in one hour Generates transfer maps automatically in real time Saves time by efficient trans
69. ns C Maintain survey history Output Options Output Files f Run specitic Source Plate specific affected by processing order Transter Reports Save transter reports as file type iY xm Ie csv TXT Format Header 6 Body 12 Footer 3 Directory lt DEFAULT gt Filename DEFAULT Survey Reports Save survey reports as file type E xm csv FP TXT Format DEFAULT Directory DEFAULT Filename DEFAULT Figure 4 20 Options Window Note The options settings are similar to the Preferences Options window but apply only to the displayed protocol Echo Dose Response User Manual 79 Software Reference 80 4 5 1 Survey History The Survey Historytunction stores survey data for source plates When the source plate is returned to the Echo liquid handler the Dose Response software recalls and uses the stored survey data instead of resurveying the plate This feature can reduce processing time during a protocol run particularly if the source plate is swapped several times during a protocol run You can specity the length of time that the survey data is retained from a few minutes to never resurvey The software stores the survey data by plate barcode therefore the data is independent of the protocol and can be used repeatedly in different protocols until the expiration time Note Various factors can affect the content of the source wells such as DMSO concentration humidi
70. nse User Manual Software Operation 3 2 4 Create destination plate template 1 Click the Add Destination button in the P ate Design window to start the Destination Plate Designer wizard The destination plate template is similar to the intermediate plate template You can assign controls to specific wells assign DMSO only wells or block certain wells from being included in the compound transfer 2 Select the destination plate from the Destination Plate Type menu For example select Greiner_384PS_ 781096 Destination Plate Designer step 1 Design the destination plate template select the wells in the microplate and then click on one of the buttons in the Legend to define the type for the selected wells in the destination plate template When finished click on Next Destination Plate Type Laboyts_384P P_StdW Labeyte 384PP Std Aurora 3456COC_00017829 Greiner e Sg Labeyte_1525C0C_LoBase_ Dest Labeyte 384C0C LV Dest Laboyte 384PP StdW Dest Figure 3 17 Destination plate type menu Note Since the destination plate will be receiving the liquid transfer you have a broader range of plate types to choose 3 Click the Next button The next screen provides fields to calculate the dose response curve The data you enter will detine the layout of the final assay plate 4 Enter the number of data points in the Dose Response Curve box see Figure 3 19 Specify the Vumber of data points per curve 13 12 pt curve plus
71. numbers the wells from the Starting Number Does not affect control or DMSO wells Direction box Specities the direction to replicate the wells All selected components compound control DMSO are replicated but only the compound wells are numbered Across Replicates the selected well horizontally in all available wells in the plate JS TEERTEERERREN I E E S Step 2 Choose a name for the source plate template Provides a name for the source plate template ax Step 2 Choose a name for the source plate template The name of the template must be unique within the system The plate preview of the plate is also shown below Template Name lt lt Suggestion Source Plate Plate Type Labcyte_384PP_StdV Plate Preview Dit ds PE OAAIIAAAAAAVOCIVDONAN OOOOOOOOOOOOOO O OOO0000 OOOOOOOOOOOOOOO OO0000 0000000800808 8E 000000 e a e gt SaVVLOVOVVALVLAAN aOSOVCOVVVVVAA _ M B Q Q E 1900 aaa aaa Ona 56665466 Cancel lt lt lt Previous Figure 4 5 Source Plate Designer step 2 Echo Dose Response User Manual 57 Software Reference 4 2 2 Intermediate Plate Designer The Add Intermediate button begins the ntermediate Plate Designer wizard in a new window This wizard help you prepare a template for additional compound concentrations Step 1 Select wells to reserve or bulk fill with DMSO only Defines the intermediate plate type Assigns wells to DMSO or bloc
72. ompounds against the target antigen Compounds showing significant activity against the antigen are selected for the next step ODO DOO OOOO CODOS OOODOOCODOODO O0O0O0OOOODOODOO ODPODOOOOCOODOO SOELPEVLEILSLLLL OOO OOO DOS OOODOOOO0ODOO Figure 1 1 Screening plate 2 Perform serial dilutions of the selected compounds See Figure 1 2 Dilutions are typically calculated at logarithmic levels for example 100 mM 10 mM 1 mM 0 1 mM 0 01 mM Each dilution represents a point in the dose response curve 3 Transfer compound dilutions to assay plates for testing 1 uL 2 mL DMSO backfill in every other well Figure 1 2 Manual dilution and transfer 4 Test the compound dilutions using techniques such as fluorescence or spectrophotometry and graph the compound concentration against enzyme activity Echo Dose Response User Manual 3 Introduction Steps 2 and 3 are the most time consuming and tedious steps for most laboratories They can also be quite expensive if the dilution step requires large quantities of compound and diluting agent such as DMSO This is the area where the Echo Dose Response software can help The Echo Dose Response software together with the Echo 55X liquid handler can prepare compound concentrations for dose response analyses by transferring nanoliter volumes of compounds directly into assay plates The Echo system can also be used to prepare an intermediate source plate if additional starti
73. our source plate and transfer them into your final assay plates to generate all the compound concentrations necessary to create an 1C50 curve If you need to expand the range of the IC50 curve the software provides an intermediate plate template to create additional source concentrations The steps to create the sample transfer protocol are listed below and explained in detail in the subsequent sections 1 Start a new protocol Enter the name and description 2 Create the source plate template Assign compound concentration 3 Create the intermediate plate template Create compound intermediate concentrations 4 Create the destination plate template Enter the number of data points compound transfer volume assay volume number of replicates and fill direction 5 Specity the order of transferring compound 6 Save the protocol Echo Dose Response User Manual 19 Software Operation 3 2 1 Start a new protocol 1 Click the new protocol icon L in the toolbar at the top of the screen or select New from the F e menu to open a new Protocol template The new protocol template opens in the Main Window The Plate Design window is displayed in the left pane and workflow diagram in the right pane Labcyte Echo Dose Response Application E a Ol x File Protocol Tools Help New LYS aob Protocol gt Protocol a Tempiaig Fai Des opos z LABCYTE D scription Create Source Plate Plate Plate templates in this
74. ource Plates 1 Partial plates as defined by a pick list Figure 4 14 Run Options window Control Buttons Connect Allows connection to the Echo liquid handler that is selected in the Instrument field When the software is connected it is online Disconnect Breaks the connection to the Echo liquid handler When the software is disconnected it is offline Simulate Opens a mock run window to simulate the protocol on the computer before performing the actual transfer Full plates Plate templates defined in the transfer protocol This is the default setting The setting Number of Full Source plates allows you to process more than one source plate in the protocol run See Example 2 in the Appendix Partial plates Plate data imported from a pick list The setting Remove Duplicate Entries instructs the software to remove duplicate wells that contain the same compound from the processing list The setting Pre process pick list in the order specified instructs the software to determine the source plate order in the pick list before the run Echo Dose Response User Manual 65 Software Reference 4 3 1 Run Status The Aun Status window is a dynamic window that provides control buttons to run the protocol It also contains various status windows to show the progress of the compound transfer the state of the Echo liquid handler and the final well positions and volume of the transferred fluid Screening i
75. pecific affected by processing order Transter Reports Save transter reports as file type M xm M csv TXT Format Header 6 Body 1 Footer 4 Directory EAiLabcrte Echo Reports NameS RunDates lnstrSN SRunT ionlD Survey Reports Save survey reports as file type M xme M csv TXT Format Header 6 Body 8 Footer 4 Directory C Labeyte Echo Reports Fiename Names S5RunDateSSinstr WNs SRunT PlateNameSiS5r Figure 2 13 Preferences Options screen 14 2 Select Maintain survey history if you want to re use the current plate survey for more than one transfer operation for a specified period of time For more information go to Survey History on page 80 3 Select Run specific or Plate specific output files Run specific Store all transfer report data in one file Plate specific Store the transfer report data in separate files based on source or destination plates depending on which process order you specified in the P ate Design window 4 Customize transfer and survey reports File type Select XML CSV and or TXT file formats Format Customize the transfer report header body and footer Directory Change the directory location for the transfer report Filename Change the file name for the transfer report These options will be used as default settings for all transfer protocols If you want to change the options for an individual protocol use the Option
76. protocol ieee Design Protocc m Plate Template Actions Wi n d ow source plate types control DMSO Add Source Template Workflow Diagram Process plates in this protocol Automatic Protocol defines the optimum sequence Source plate centric Minimize source plate swapping Destination plate centric Minimize destination plate swapping Figure 3 1 Main Window The new protocol is temporarily labeled Protocol 1 until you save it 2 Enter the name and description Type the name and description of the protocol in the text fields The description should have sufficient information to serve as a reminder the next time you open this protocol Name 12 pt assay with 3 compounds Description Source plate with 3 compounds intermediate dilution plate with 2 dilutions per compound DMSO backfill Figure 3 2 Name and description of new protocol 20 3 2 2 Create the source plate template The Plate Template Actions box contains the function keys to design the plate templates and add them to the protocol Plate templates in this protocol i Plate Template Action Directory tree of Protocol Plate Template Actions plate templates El Source created for this Source Plate protocol Functions keys to manage templates Information on 3 Thereis 1 source concentration per compound in this protocol source concentrations Figure 3 3 Plate template tree a
77. r countries Pentium Ill and Pentium 4 are trademarks or registered trademarks of Intel Corporation or its subsidiaries in the United States and other countries All other registered or unregistered trademarks used herein are the exclusive property of their respective holders For research purposes only not for use in diagnostics The Echo liquid handler is covered by one or more of the following patents 6 416 164 6 548 308 6 603 118 6 612 686 6 642 061 6 666 541 6 707 038 6 710 335 6 746 104 6 802 593 6 808 934 6 809 315 6 849 423 6 855 925 6 869 551 6 893 836 6 893 115 6 916 083 6 932 097 6 938 987 6 938 995 6 991 917 7 070 260 7 090 333 European Patent EP 1337325 Additional patents pending in the United States and other countries Contents Prora EE E EEE V MAA aa V Software Requirements c ccccccccccssscsecsccsecsessecsecsessesseecseceessassaecseseesseseaecatsaeesaeeaesass V Mamuak Organiza UO eeN V Manual Convention csn vi Hi e E EE 1 AA e o er eee ene eee ee 1 1 2 What is a dose response CUIVE c ccssccccsecscessecsecssecseseecsessessessessessaeeauscatenaeegs 2 1 3 How does the Echo Dose Response software fit iN ooooiccnconiccoccncncncnncnonos 3 1 4 What are the benelli Fenicion neinna nana e A AAR 5 Getting Started iran a a ORC 6 Dsl SONAS T a E E AINNE 6 Z2 DOCUMENTAU o va i ttt eter ote 8 Ls MON eterna 10 e see ee ee ee rere ee eo ae ne ee eee ee ere 12 Dealt AAPP a 12 DAL SET
78. r the following information in the Dose Response Curve box right dialog box Specify the Number of data points per curve 14 Enter the Assay Volume for all destination wells 20 uL e Enter the following data in the Data Point Curve table lett dialog box Source concentration and control name trom the drop down menus Transfer volume for each data point Echo Dose Response User Manual 93 Appendix 94 The software calculates the backfill volume of DMSO and final compound concentration for each data point acktill Transfer Volume nL Volume nl 2 sooecom sf sol o o os oo INESIS O ROMINA IESO CO o INESIS IOMA ROMINA IESO SSC OT 7 es INIA EOI ET assess reo o swen soi al amo fa soncorw IAE IRMA E TO me lf sof ofS o w rene ll 0 of o Figure A 10 Dose response data table Note This example uses a 12 pt curve plus 2 controls for a total of 14 data points therefore the option to include controls is selected f Return to the Dose Response Curve box and enter the DMSO backfill volumes Select the Automatic DMSO Backfill option Select the timing for the backfill Last after compound transfer The software will calculate the volume of backfill needed and add it to the Data Point Curve table Select Maximum Backfill Volume The default value is the highest transter volume which is 75 nL in this protocol Select Backfill offline for volumes greater than equal to
79. re 4 7 Source Plate Designer Step 2 Step 2 Fields How many intermediate concentrations would you like Number of intermediate concentration fields to be listed in the concentration table Source Concentration Drop down list of source concentrations previously defined in the source plate template Transfer Volume Volume of source compound in nanoliters to be transferred from the source plate Bulk Fill Volume Volume of DMSO in microliters needed to dilute the source compound New Concentration Concentration of the intermediate compound The Echo Dose Response software automatically calculates this concentration 60 Note The range of new concentrations created depends on the working range of the source plate that is used The plate with the widest range is Labcyte 384PP StdV which has a working range from 20 uL to 50 pL Working ranges on other source plates can be found at www labc te com click Products menu select Consumables and open the Web page for Echo qualified microplates Step 3 Choose a name for the intermediate plate template Provides a name for the Intermediate Plate Intermediate Plate Designer Axl Step 3 Choose a name for the intermediate plate template The name of the template must be unique within the system Template Name Intemediate Dilutions Plate with DMSO Plate Type Labcyte_384PP_StdV Plate Preview 1 2 3 lt 5 6 7 e 9 J10112103 0 405 frefs7fs8 1920 21 e2 23 24
80. requirements with one source plate Depending on the plate layout of your destination plate how many empty wells remain after a run the ability to run multiple source plates in a single run can make use of the empty wells and significantly reduce the number of intermediate and destination plates that are needed Echo Dose Response User Manual
81. ry C Labeyte Echo Reparts Filename ID e F Save Cancel Figure 4 29 Preferences Options Window Echo Dose Response User Manual 87 Appendix Example 1 12 pt curve with controls Plan Curve Data This example is a 12 pt curve with controls Starting concentrations of your compounds Layout for the source plate One source plate template containing compounds controls and DMSO Layout for the intermediate plate One intermediate plate with two dilutions from stock 1 100 250 nL 25 uL 1 10 000 250 nL 25 uL Final assay volume 20 uL Number of points needed for IC50 graph Set up the transfer protocol 1 Start a new protocol Click the new protocol icon L or select New from the Fi e menu to open a new Protoco window 2 Type the name and description optional of the protocol in the Plate Design window Plate Design Options Name 12 point curve Description Uses 5mM source concentration with final assay volume set to 0uL This example incorporates the controls and DMSO backfill in the destination plate Figure A 1 Protocol name and description 3 Design the source plate template using the following steps a Click the Add Source button in the P ate Design window to open the source plate designer window b Select the plate type of the source plate for example Labcyte 384PP StdV c Select the wells that must be reserved for the controls and DMSO Echo Do
82. s as defined by a pick list Figure 3 30 Run Options window offline 4 Click the Play button lower right corner of the window to start the simulation run If the protocol is complex or contains several plates the software will require extra time to load the information To simulate a transfer run the software will display prompts to insert the source intermediate and destination plates into the Echo liquid handler You can respond to these prompts or allow the software to respond to the next logical step after a 5 second delay Insert Source Plate 3 sec l i ep Please insert Source Plate Labcyte _384 P _Stdv Figure 3 31 Plate prompt during transfer simulation During the simulation you can click Skip Animation or Cancel Echo Dose Response User Manual 41 Software Operation Transfer completed red L Transfer in progress blue Original conc Original 5 Click OK when the simulation is complete The following figures show a simulation in progress and completed Number of plates required 16 compound assay edr Simula 16 compound assay edr 4 bx Protocol E e Echo Protocols 16 compound assay edr Source Plates 1 Intermediate Plates 1 E Destination Plates 1 Plates In progress Sessions 2 Current Session 2 Current Transfer Map 1 Transfer from Intermediate Dilutions Plate 01 2 Par 7 Transfer to Destination Plate 01 3 E Compound Empty ves
83. s available in the Echo Dose Response software It contains screen descriptions and procedures You can view online Help by using one of the following methods in the software e Click the Help icon a or Help menu in the software toolbar to view the complete Help file Labcyte Echo Dose Response Contents Index Search About Figure 2 5 Access to Help from the Toolbar e Click the Help icon 2 in the upper right corner of individual software screens to view the Help page for that screen Export Plates Ex C Aurora_3456C0C_ 0001 78 29 C Greiner_1536HibasePS5_78 4096 C Greiner_384P5_ 781096 C Coming_1536C0C_HiBase_Dest C Labcyte_1536COC_LoBase_Dest Figure 2 6 Access to Help from the software screen Press the F1 key on the computer keyboard to view the Help page for the displayed screen that does not have a Help icon Quick Start Guide The Echo Dose Response Quick Start Guide describes the capabilities of the Echo Dose Response software and takes you through a sample protocol _ Echo Dose Response Software The Echo Dose Response software enables the user to do the following Choose source wells Define layout of source and destination plates Specify the number of points in the dose response curve Choose the concentration and volume of the compounds to be transferred Figure 2 7 Quick Start Guide The Quick Start Guide can be found in the Echo Dose
84. s tab See Preferences on page 87 or Protocol Options on page 79 for details I The software is set up You are ready to create and run a transfer protocol Echo Dose Response User Manual 15 Getting Started 2 5 Software Shutdown You can exit the Echo Dose Response software in the following ways e Open the F e menu and select Exit Protocol Tools Help Connect to Instrument New Ctrl Open Ctrl 0 Figure 2 14 Exit function in the File menu e Click the Close icon in the software window a p x Figure 2 15 Close Window icon The software will prompt you to save your protocol before it shuts down 16 3 Software Operation The best way to describe the features of the Echo Dose Response software is to take you through the steps to create and execute a transfer protocol What is a transfer protocol In the Echo Dose Response software the transfer protocol is a set of plate templates and transter options that help you design the dose response curves up to 24 point in the assay plates The software automatically creates the optimal transfer sequence that is sent to the Echo liquid handler to execute the direct compound transfer to assay plates After the transfer is complete you can retrieve custom reports that document the transfer run and provide well data tor sample tracking Sample transfer protocol The sample transfer protocol in this chapter uses a single column of compound to illustra
85. s the status of each plate as it progresses through the transfer protocol The Legend interprets the symbols used in the status windows Legend Complete In Process m Fending Enor Echo Dose Response User Manual 67 Software Reference SOURCE PLATES DESTINATION PLATES Source Plate 01 Plate 7 Source Plate 02 Plate 3 vy E Source Plate 03 Plate BM Source Plate 04 Plate 4 Source Plate 05 Plate5 Transfer Map window Shows the details of each well to well transfer TRANSFER MAP Source Well Dest Well Transfer Volume 122 AG 25 System Status window Shows the status of the Echo liquid handler during the transfer run If a problem occurs the alert will appear on this screen 68 Transfer Status Shows a progress bar of the transfer protocol as well as percent completion and elapsed time This section also provides a text window that displays all the actions that occur during the run Echo Dose Response User Manual 69 Software Reference 4 3 2 Simulator The simulator is an animation of fluid transfer for the selected protocol The simulation is interactive and allows you to respond to a prompt box to insert and remove plates Since this is a simulation the prompt box appears for only 5 seconds then the software automatically select the first available plate and continues to the next step The protocol example in Figure 4 16 is a simple source
86. se Response User Manual 88 This example uses a source plate that contains compound controls and DMSO for backfilling therefore some of the wells must be reserved before assigning compounds Select the well that contains the 1 2 3 4 5 fe 7 s 9 10 11 12 13 first compound A1 gt AOOOOOOOOOOOO0O BEECK aa na SNA na h Click the Compound Conc Button to open the Assign Compound Concentration tor Selection window Name optional D E 5 Cone Po so0 mm G El Pa Figure A 2 Assign Compound Concentration Enter the compound concentration by typing the number or using the arrow keys Modity the concentration units if necessary Leave the compound name blank and click OK Enter the compound base name and starting number in the Rep cate Pattern box Replicate Pattem Select the wells to copy Enter the base compound name and starting number click the direction to copy the wells and then click the button below Base Name Kinase Starting Number 1 Replicate Selected Pattem Direction Across f Down Undo Replicate Pattem Rename Selection Figure A 3 Replicate selected pattern Select the replicate direction for the compounds Down Echo Dose Response User Manual 89 Appendix 90 Click Replicate Selected Pattern The compound concentration 5 mM will be replicated down the plate into all available wells
87. te a simple compound transfer This protocol will use the original compound concentration in a source plate as well as two compound dilutions in an intermediate plate to create an assay plate that can generate a 12 point IC50 curve Throughout this overview there will be references to specific topics in the Software Reference chapter beginning on page 50 which can provide additional details The steps to creating and executing a transfer are listed 1 Plan the Curve Data provided by the user to create the protocol 2 Set up the Transfer Protocol created by the user in the Echo Dose response software 3 Set Up Plates performed by the user before running the protocol 4 Specify the order of transferring and diluting compound performed by the Echo Dose response software and Echo 55X liquid handler 5 Run the Protocol performed by the Echo Dose response software and Echo 55X liquid handler 6 Manage Transfer Data generated by the Echo Dose response software based on user Settings Each step contains screen descriptions and procedures By the end of this section you will have a good basic understanding of the Echo Dose Response software Note After you have read through the protocol in this section review the sample protocol in the Appendix It describes additional software options that may be more applicable to your needs Echo Dose Response User Manual 17 18 Software Operation 3 1 Plan the Curve Data Be
88. termediate Dilutions Plate E Destination Destination Plate PolZlEt alee rfofoley gt GOOQOOHOOOOLHOOOO _ POOQOOGOOOOLOOOOO GOOQOOOOHOOSOOOSOO GOOQOGOGOOOO OHOOOOO GOGQOOGOOOO OOOOOO GOOQOOQHOOO OHOOOO GOGQOOGHOOOOHOOOO _ POOQOOGOOOOHOOOO OOOOOQGOOSHQOOHOOSOOO GOOQOOQOOOOOHOHOOOO GOGQOOQGOOOO OHOOOO POGQOOGHOOOOHOOOO POOQOOQGOOOOHOOOO GOOOOOOGQOOHOOSOOO_ dOSOGOGOGGOGODALS PHQOOQOOQOOQOOOOOOOOOE MHQOOOOOOOOOOOOOOOE GOOOOESGOOEOOE OOOO OE GOOQOOOESGOOEOEOOOEOOS Els OO0900000000000001 OC090000000000000Hl OCO0O0O00000000000001 O0O00000000000000 OO000000000000000k There are 3 source concentrations per compound in this protocol Figure A 7 Preview of source plate template 4 Create the intermediate plate template a Click the Add Intermediate button in the P ate Design window to open the ntermediate Plate Designer window b Select the plate type of the intermediate plate Step 1 Select wells to reserve or bulk fill with DMSO only Click on either the column heading or the row heading to select the wells in the intermediate plate C automatic processing or on Bulk Fill DMSO to fill those wells with only DMSO as part of a bulk fill o Intermediate Plate Type ee 304PP aan ES Figure A 8 Select i oia slate in Note The intermediate plate must be an Echo qualified source plate since liquid will be transferred out of this plate Select the
89. tination Plate Type Labcyte 384PP_StdV p gt Transfer from 01 to E8 Skip Animation Play Figure 3 32 Simulation in progress 16 compound assay edr Simula 16 compound assay edr db x Source Plate Type Protocol C Labcyte Echo Protocols 16 compound assay edr fLaboyte_384PP_StaV Source Plates 1 sas aaa a a eae eee eae DOS ooo oO ntermediate Plates 1 9004040000000 000 00 a DDED Destination Plates 1 OOOO ODO OOOO OO OOOOOOO dd BOSOSOSGOGOGOSSHSSHS3HSH H ees j E T ED HOOSCSOOOOOODOODOOODODODO0OD Current Session 2 S OOOCOOOO OOOO ODO DOOOO OO OOO HOOCOCOODOoDoOcCODO OO OOOOdOd Current Transfer Map 0 HNOCOCOCOCO O CODO O OOOO OOOO OOOO OC OO OOD OOOO xj ransfer from Intermediate Dilutions Plate 01 2 OOOO OOO OSOOoS ae AHOCOCOCOODOCOOOOOO0O ransferto Destination Plate 01 3 MOOCOCOCOCODOCOO OOO OOOOOOOE MOOSSSSSSS605 ascanascacoc s L Protocol simulation Complete nao Dilution 1 Dilution 2 3 Soe 2000 QOG QOG 200 Soe OOG 20500 QOG 200 250 ae Soe 250 QOG conc TDT Transfer from W16 to B16 with nc o a Dilution 2 with DMSO backfill Skip Animation Piv ose backfill 42 Dilution 1 with DMSO backfill Figure 3 33 Simulation completed 3 5 Set up Plates Use the design of the plate templates and the number of plates specified in the simulation to set up the plates for your compound transfer 1 Prepare an Echo q
90. to destination plate transfer for a 5 pt dose response curve The components of the simulator window are described below 5 pointdoseresponse assay edr Simulator C 1L ponse assay edr qb x Source Plate Type Protocol CA Labcyte Echo Protocols Source Plates I E E E A CA iD E E 5 E A ada ae ood ie Sessions 1 E Destination Plates 5 1 Curent Session 1 1 Curent Transfer Map Transter from Source Plate 1 Well Color Legend Compound Empty Transfer to Destination Plate 03 4 EN El Ea El El El El O E EA o EI E El La Prefetch Plates ID Name Type Wells Barcode 1 Source Platel Labcyte_384PP StdW 384 2 Destination Plate 01 Labcyte_384PP_StdW_Dest 384 3 Destination Plate 02 Labcyte_384PP_StdW _Dest 384 4 Destination Plate 03 Labcyte_384PP_StdV_Dest 384 Transfer from Fito R10 Play Skip Animation Figure 4 16 Simulator Window The transfer animation lower right corner of the window simulates the movement of the destination plate green as it receives liquid from the source plate Each source to destination well transfer is documented below the plates Transfer from F7 to E11 70 Control Buttons Skip Animation bypasses the animation of the current step and shows the completed step Play begins the simulated compound transfer Close closes the simulation window If a simulation is in pro
91. ty and exposure time during the transfer run therefore consider these factors when you select an expiration time Read the Labcyte application note on how to calculate TTL The Survey History function is located in the Options window Options Protocol Options Surveys C Re Survey every source plate Figure 4 21 Survey option Control Buttons e Re survey every source plate Default setting that automatically surveys the source plate every time the protocol is run e Maintain Survey History Expiration button opens the Survey Data Expiration dialog box Survey Data Expiration Survey Expiration Time Never expires f User specified expiration time 0 minutes OF 000 00 00 Days Hours Minutes Figure 4 22 Survey Data Expiration Control Buttons e Never expires Existing survey data is always re used e User specified expiration time Existing survey data is re used for the time that is specified in m nutes or days hours minutes Notes e lfastored survey does not cover the plate area that is required by the protocol for example some of the wells were empty when the plate was originally surveyed but then filled later on the software will survey the missing wells and add this data to the stored survey data e The stored survey data is instrument specific For example if a source plate is surveyed in instrument A the stored survey data cannot be used in instrument B e Althoug
92. ualitied source plate with the compounds you will be transferring Manually pipet Compound 1 in well A1 Compound 2 in well B1 and so on as shown in the source plate template Figure 3 34 Preparing source plate 2 Prepare an intermediate plate Manually pipet compound dilutions and DMSO into the assigned wells 3 Collect the number of destination plates that are specitied in the simulation to receive the final compound concentrations When your plates are ready for the liquid transfer continue to the next section to run the transfer protocol Echo Dose Response User Manual 43 Software Operation 44 3 6 Run the Protocol After you have created a transfer protocol and tested it in the software simulator you are ready to run the protocol on an Echo liquid handler system 1 Click the Run icon or select Run from the Protocol Protoco menu Error Check 2 Select Full plates or Partial plates Bun Ciir Le Full plates Run the source plate exactly as it was defined in the Source Plate Designer Specity how many compound source plates will be used in this run Partial plates Import and select a pick list Determine if you want to use the options Remove Duplicate Entries and Pre process pick list in order specified Run Options Instrument fcHo1 hai Connect Se al Number E55X 0791 Status Available Normal Protocol Name CC Labeyte Echo Protocols 10 pt_assay_with_backfill_v12 edr Ru
93. umentscreen Connect to Instrument Labcyte Echo Dose Response Instrument E55x 06391 E55 0690 k Type or select the name of an Echo instrument and click on Connect Work Offine Connect Cancel Figure 2 2 Connect to Instrument screen 4 Select one of the following start options Connect Select an Echo liquid handler from the nstrument menu and click Connect The Main Window opens with the connection status instrument information at the bottom of the screen This option enables you to execute the compound transfer as soon as the transfer protocol is ready See Figure 2 3 BW Labcyte Echo Dose Response File Tools Help Dae a gt Connected To ECHO 393 S N ES5354 0790 Ver 2 1 4 Figure 2 3 Dose Response software connected to Echo instrument Work Offline Click Work Offline The Main Window opens with the connection status offline at the bottom of the screen See Figure 2 4 This option enables you to create a transfer protocol and run the transfer simulation without connecting to the Echo liquid handler Labcyte Echo Dose Response File Tools Help O0SH4 0 Figure 2 4 Echo Dose Response software offline Echo Dose Response User Manual 7 Getting Started 2 2 Documentation In addition to this manual the Echo Dose Response software comes with the following useful documents Online Help e Quick Start Guide C50 Curve Calculator Online Help Online Help i
94. umn where The left edge of the part will be defined as the two 12 7 mm areas as measured from the corners as specified in ANSI SBS 1 2004 internet link Valid range 0 0 to 128 0 mm The items marked A G are measurements that need to be taken by hand or filled in from the manufacturer s specifications These dimensions and further details on their definitions can be found at Atto www sbsoniine com msdc approved php 76 A1 Y Offset B X Center Spacing C Y Center Spacing D Plate Height E Flange Height F Well Width G Well Capacity H Distance trom top outside edge to center of first row where The top edge of the part will be detined as the two 12 7 mm areas as measured from the corners as specitied in ANSI SBS 1 2004 internet link Valid range 0 0 to 86 0 mm Column spacing Each following row column shall be an additional X Y mm in distance from the top left outside edge of the plate as specified in ANSI SBS 4 2004 internet link Valid range 0 05 to 9 0 mm Row spacing Each following row column shall be an additional X Y mm in distance from the top left outside edge of the plate as specified in ANSI SBS 4 2004 internet link Valid range 0 05 to 9 0 mm 0 The overall height of the plate as specified in ANSI SBS 2 2004 internet link Valid range 6 5 to 4 5 mm The height of the flange skirt as specified in ANSI SBS 3 2004 internet link Va
95. ve finished entering the curve data it should look similar to the following figure Please fill out the table below in its entirety Source Volume Backfill Volume Assa co ra Concentration easiness a mr uni a men f 75 some ls Figure 3 26 Dose response data point window 8 Click the Next button and enter the destination template name Destination Plate 9 Click the Finish button to return to the P ate Design window The destination plate definition is complete 3 2 5 Specify order of transferring compound The section Process Plates in this protoco box is at the bottom of the Plate Design window The plate processing order depends on your priorities Select one of the following options Automatic The software selects the most efficient order of plate handling This is the default setting and may be fine for most protocols Source plate centric The software minimizes the frequency of swapping the source plate This option is useful if you need to complete the processing of the source plate and return it to storage Destination plate centric The software minimizes the trequency of changing the destination plate This option is best if you need to complete the processing of the destination plate and put it in environmentally controlled plate storage Process plates in this protocol Automatic Protocol defines the optimum sequence Source plate centric Minimize source plate swapping Destination plate centric Minimize
96. y Note If you receive a connection error Host refused connection Verify address and port number then the Echo liquid handler is turned off Turn on the Echo liquid handler wait for the green ready light to appear and then click Connect again Full plates This option allows you to run u plates as defined by the protocol you created The setting Number of Full Source Plates allows you to process multiple source plates in a single run See protocol example 2 in the Appendix Run Protocal Full plates as defined in protocol Number of Full Source Plates 1H Partial plates as defined by a pick list Partial plates This option allows you to run partial plates trom an imported pick list When you select the Partia plates option an Import button is displayed to choose a CSV formatted list of plates and wells to process You also have the following optional settings Y Remove duplicate entries e Pre process pick list in order specified Remove Duplicate Entries The software ignores duplicate wells that contain the same compound removes duplicates from the processing list Pre process pick list in the order specified The software pre processes the pick list to determine the placement of all compounds n advance of the run During the run the software prompts you for the specific source plate needed Please insert source plate Plate Name Source Plate D1 Plate Type Labcyte 384PP StdW

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