Home

BD CSampler™ Software User Guide

1. Rn ennai ee mJ mMm Joj o O Plot 2 FSC H IC Molume Events of This MeanF CVFSC H MedianF R1 Plot 4 FL2 H CH Bo ET w ara i ewa DF 100 0 Plot 5 FL3 H KE GE al v mMm bs I l I o ee fe 0 oj Mol Plot 7 FSC H SSC H Plot 3 FLI H C Nolume Events Pe of This 1 MeanF CVFLI H MedianF ba BE EL OE GE Plot 8 FL4 H Thisklot JO O Plot 9 FSC A SSC A M2 Mell a ty Plot 10 FL1 A VE Plot 11 FL2 A fu Plot 12 FL3 A on l EET ETT TE a a z REE ER J Nolume Fvents Pa of This 1 Mean F 1 CV FI 2 H Median F Add rows to your master statistics Select cells from the Sample Selector and Statistics Column Selector to build your table _ table by selecting samples Plot 1 FSC H SSC H Plot 3 FL1 H iew Addto Ri DE EE ae MS Preview Table Sample Name All Samples A03 Spk after unity 562 963 55 414 414 02 1 81 2 402 50 301 826 36 1 49 177 99 131 7 aot Bpk initia ji 562 708 27 414 372 89 180 2 402 10 301 451 04 1 50 178 37 129 87 S oo hee 562 003 43 413 560 84 1 81 2 397 07 303 343 64 1 52 175 03 128 54 Z porn l 580 276 78 521 627 05 1 44 2 170 24 301 904 00 1 32 234 05 159 1 C M A038pkafter unity BO4 Spk verifieationrun 579 43
2. ccccceeeeseeeeeeeeeeeeeseeeeaeeeseees 34 Fig re PA View ora POl sessiossa 35 Figure 4 13 New Density and Histogram Plots ccccccseccseeeseeeeeeeeeeeeeeeeeeeeeeeseeeneeeaeeeneeenaes 36 Figure 4 14 Set Plot Specs Dialog BOx rrrrrarrrnnnnerrnnnrvnnnennnnrrnnnnennnnrnnnnnennnnennansennnnennnsennnnene 36 Figure 4 15 Before and After Changing Events Displayed cccscccceeceseeeeeeeeseeeseeeseeeesaees 37 Figure 4 16 Events Display Settings Dialog Box rrrarrrnnnrrrrarrvrnnnenrnnenrnnnennnnennnnnennnnennnnnennnnen 38 Figure 4 17 Plot with Events Display Settings Applied rrrarrrrnnnennanrnrnnnrrnnnrrrnnrernnnenrnnnennnnen 38 Figure 4 18 Using Polygonal Gating TOON ai ndocnwstntececasndonnssincnecseaeieansstniocasaeeienisindeegssehteenssieiens 39 Figure 4 19 Using the Vertical Marker rrranrnnnnennnroranrrrnnnennnrnnnnennnnennnnnnanennnnennsennnnennnnennsnnnne 40 iv 7820099 01 Rev 0 Figure 4 20 Using the Horizontal Marker c ccccceeccseeeceeeeceeeeseeeeeeeseueeseeeseueeseeeeseeesaeeesaees 40 Figure 4 21 Selecting a Gating Option rnrrnnannnnnnnenrannnnnnnenranrnnnnnrnnnnrnrnnsennnnennansennnnennnssnnnnsen 41 Figure 4 22 Plot Galed to Include PI Kaase 42 Figure 4 23 First Gate for Creating Nested Gates Drawn in Plot 4 rrrrnrnnrrnnnnnrvernnvrrnnnnerennne 42 Figure 4 24 Applying the Parent Gate to Plot 5 rrrrnnrrnnnrenrnnrrnnnnennnnennnnnennnnenrannennn
3. ccccceecccseeeeceeeeeseeeeseeeeeeeeeeseeeeeaaees 118 Ea Optical Filler Placement 119 E 5 Selectable Laser Application Examples cccccccseccccseeeceseeseseeeneesessaeeenseeensaees 122 E 5 1 2 Blue 2 Red Configuration Examples ccccccsececsseeeceeeeeeeeeseeeessaeeesees 122 E 5 2 4 Blue Configuration Examples ccccccccccsseeeceeceseeeeeceeeseeeeeseusesseeesees 124 APPENDIX F ENHANCED ANALYSIS FEATURES swsisticseeieieecrtiaievintividectviadaeietieideartiontes 127 Fi Greating a LIVe Galen aa 127 F 2 Renaming Plots and Regions rrnnrnnnrnnnrnnnrnnnrnnnrnnnrnnnrnnnennnrnnnennnennnennnrnnnennnrnnsennee 128 F 3 Coloring Events ina Region cccccceeccceececeeeeeceeeeceeeeeseeeeceueessaseeseueesseesseeesaaees 129 F 4 Creating Publication Quality Images of Plots rrrnrrrnrnnrrrrnnnnrnrnnnrenrnnrrnnnnrrennnnsnen 130 F 5 Analyzing Batches of Samples c ccccccsececceeesceeeeccecesceseeceeeeccesecceseesoeeesceses 130 F 5 1 Viewing the Batch Analysis Tab rrarennnrnnnrvnnnvnnrvnnrnnnrvnnennnennnennnennnennnennnen 130 R52 RUINNNGE BECNANANSE avsender danne 131 pe EE gt 010 011 01 DEV RE 132 APPENDIX G FCS KEYWORDS se 133 APPENDIX H EXAMPEE GSV FILE suges 136 7820099 01 Rev 0 iii BD CSampler Software User Guide TABLE OF FIGURES Figure 1 1 BD CSampler Accessory Kit rrrrnnrnnnnnnnnnnnenrnnrrnnnnennanennnnnennnnennnnnnnnnnennnnsnnnnennusennnn
4. ccccccccsececseeeeseeeceecesseeeesesesseeeeseeeesseeeseeeesseeeess 61 Fiqure 4 55 OMe lait FOS TE aa 62 Table 5 1 Auto Collect Tab Controls se 65 Figure 5 2 Plate Type Drop Down Menu rrnanennnrnnnrnnnrnnnrnnnrnnnrnnnrnnnennnennnennnennnennsennnennnennsennee 67 Figur 5 3 Plate Name Field srecetina anaE ERICEK a EEEREN EEE 67 Figure 5 4 The Auto Collect Tab Displaying Two Data Sets ccccccccsseeeeseeeeseeeeeseeeeeeeeeeas 68 Figure 5 5 Selected Wells in the Auto Collect Tab rrrrnrnrnnnnnnvnnnnnrnnnnvevernnvrvnnnnernnnnvevennneennnne 69 Figure 5 6 Samples in a Set Are the Same Color ccccccscccceeeeeeeeeeeeeeeeeeeeeeeeeesseeeeseeeesseeeess 70 Figure 5 7 Agitate Plate ControlScan aaa eee eed es ee ane 73 Figure 5 8 Wash Settings Controls cccccccccsecccceeeeceeeeeseeeeeeecesseeeeseecesseeeeseeeesseeeseeeesaeeeeas 73 Figure 5 9 Run Direction Controls arrrrnnrrnrnnrrnnnnenranrrnnnnenrnnrnnnnnennnnennnnsennnnennansennnnennnssennnnen 74 Figure 5 10 Sample Annotation Table rrrranrrnnnrorranrrrrnnnrnrnnrnrnnnrnrnnrnrnnnnnnnnennansrnnnnennnnennnnnene 74 Fiqure 5 11 BD Csampler Run DISOlay sc 2ncatecet teeta eed ta diceied ei aliete ate adinte ote aed es 75 Figure 5 13 The BD CSampler Software Display After a Sample RUN ccecceeeeeeeeeeeees 76 Figure 5 14 Run Display Viewing Sample PIOtS cccscccesseeeeeeeeeeeeeeeeeeeeseeeeseeeeeseeeesseeeess 77
5. rrranrnnnnonnnrnnnnrnnnnnnnnrnnnnennnnennnrnnnnennnnnnnnennnnennnenn 19 3 5 Troubleshooting Validation ccc ceeccceeeeceeeeeeeeceeeeseeseeeeceeeeseeeseueeseeeeseeeseeeneeesaeeens 20 a MANUAL IDAIA ACO WES INO Nere 22 4 1 Viewing the Manual Collect Tab r rannrrnnnnnnnnnrnnnnnnnnnnnnnnnnnnnnnnnnnnrnnnnnrnnnnennnnnennnnennnn 22 4 2 Collecting Sample Data rrrnnnnrnnnnnnnnnnrnnanrnnnnnrnnanrnnnnnennnnennnnnennnnennnnernnasennanennnsennnn 25 4 2 1 Setting the Fluidics Rate 2 0 cccccceeeeceeeeeseeeeceeeesseeceseeeesaueeeseueesseeesees 26 4 2 2 Setting the Threshold esirin i aiiai aai 26 4 2 3 Assigning a Plate NPE icin trivssnonionatecrivsironinisehivcinonnnamehiveiromnametndueaneans 28 424 Naming the Plafar a a E annaa 30 4 2 5 Naming the Sample cccccccccsccecseeceseeeeceeeeeeeeeeseuceeseeeeseeeesseeeeseeeesseeesaes 30 4 2 6 Setting a Run Limit onrennnnrrnnnnrnnannnnnnnvnnannnnnnnrnnannnnnnnrnnansnnnnnrnnnnrnnnnnrnnnnsnnn 31 4 2 7 Running the SAM Ole oie scseaccoececestacenzacesencdednaceancisdancdedsacdsnansdanssetsaeweaeacnsacdae 32 4 2 8 Washing the SIP Between Samples ccccsecceeeeeeeeeeeeseeeesseeeeseeeesseeeesees 33 4 2 9 Adding New Sample Data to a BD CSampler Software File 08 33 4 2 10 Pausing Data Collection ae 34 4 3 Ending a Data Collection Session rrrrarrrrnnnernnnrrnnnnerrnnrnnnnnennanrnrnnnennnnenrnnnennnsennnn 34 Aa GMP vr 35 4 5 Changing
6. cccccccscccsseeccsseeceeeecseusecsueecseeeessueesseeeessaees 12 Figure 3 2 Select Wel A hunn 12 Figure 3 3 Run Limits Disable Run Unlimited orrrrnrrnnnrennnrnrnrernnnennnrrnnnrnnnnennnrnnnnennnnennsennnne 12 Figure 3 4 Run Limits 50000 Events cccccccccsecceceeeceeceeeecaeceeseeeeeeseeeeesseeesseaeeesseeeeesanes 13 Figure 3 5 Sample Name 8 Peak Beads cccceccccseeeceeeeeceecesseeeeceeeeseeeeeseueeseeeeseeeessneeeas 13 Figure 3 6 Select Well for 6 Peak Beads ccccceccccsecccseeeeceecesseeeeseeeesseeeeseusessaeeeseeessaeeeas 14 Figure 3 7 Run Limits 50000 Events cccccccccceecceceeeceeceeeeseeceesaeeeesseeeeesseeeeeseaeeesseeeeesaaes 14 Figure 3 8 Sample Name 6 Peak Beads ccccceccccseceeeeeeeceeceeeeeeeeeeeeseeeeeseesesaaeeeseeeessaeeeas 14 Figure 3 9 Run Limits 2 MINutes cccceccceecceeeeceececeeeceuceceueeceeeceuseseueesueessueeseeeseeessueenaees 15 Figure 3 10 Plot with Bead Doublets ccccccceccceececeeeceeeseeeeseeeseeeeseueeseesaueeseeeeseeesaeeesaees 16 Figure 3 11 Gate Applied to 8 Peak Bead Plot rrrrrrannnnnnennnnnnnnennnnnnnnrnnnnennnnnnnnrnnnnennnnennsrnnnne 16 FIGUKE 3 12 ZOOMEO VIEW OF Plassen 17 Figure 3 13 8 Peak Validation Bead Data rrraronnnnonanrrnnnennnnnnnnrrnnnennnnnnnnennnnennnrnnnnennnnennsennnne 18 Figure 3 15 Statistics Tab 8 Peak Bead Data from Successive Days ccccsseeeeeeeeeeeeeees
7. png default lower resolution image Excel compatible C eps higher resolution image for use in publication software such as Adobe Illustrator and Photoshop Excel is a registered trademark of Microsoft Corporation in the United States and or other countries Adobe Illustrator and Photoshop are registered trademarks of Adobe Systems Incorporated in the United States and or other countries Figure 4 53 Set Plot Drag and Drop Format Dialog Box NOTE The Drag and Drop Format selection is BD CSampler Software file specific and will always revert to the default of ong when a new BD CSampler Software file is created or if the current BD CSampler Software file is not saved with the eps option selected e Click on the OK button to save the settings and return to the BD CSampler Workspace 4 18 Printing Data To print selected plots and associated statistics from the Manual Collect or Analyze tab e Enable the check box in the upper left corner of one or more plots Plot 1 401 Unstained X Plot 2 404 Unstained Plot 3 401 Unstained GATE No Gating GATE No Gating GATE No Gating 10 000 12 800 40 000 51 200 200 000 400 000 600 000 300 000 Count 20 000 F 00 000 1 000 000 1 600 000 0 5 000 000 10 000 000 16 777 215 FSC A FL1 FSC A 0 o Ca SEK z sa i 100 0 100 0 429 939 3 115 800 4 72 9 162 9 Figure 4 54 Plots Selected for Printing The associated statistics are automatica
8. Figure 4 36 Rename Parameters Dialog Box e Doone of the following e Select the Sample XXX radio button where XXX refers to the current sample to apply the label to the current sample only e Select the All Samples radio button to apply the label to all samples Click OK To assign the name to the same parameter in another sample e Select another well in the sample grid e Click on an axis label in a plot and select Rename Parameters from the drop down menu e Select the name from the drop down list associated with the parameter 48 7820099 01 Rev 0 BD CSampler Software User Guide 4 11 x Rename FL1 to dl none v Rename FL to 12 none Rename FL3 to CD45 PE cy7 RLS 1 select ve RenameFl to 4 EG CD45 PE Cy7 Apply ko Sample HPB CD45 PE Cy 7 OK ae All samples Figure 4 37 Rename Parameters Dialog Box with Axis Label Drop Down List Zooming on a Plot BD CSampler Software automatically zooms the initial display of any parameter ona logarithmic scale from channel 10 to 16 7 x10 For most analyses very few events fall into channels 0 to 10 so automatic zooming saves time by reducing the number of zoom steps However take care when setting markers M regions R or polygons P that require channels lower than 10 on a zoomed plot It is recommended to unhide the first decade of data See section 4 5 to prevent events from being excluded from gated regions especially whe
9. Run Unlimited Slow Medium Fast Run with Limits t Flow Rate 14 uL min 08 Core Size 10 pm r3 wy es u E 3 Custom de MB in Ungated Sample M2 M3 Me i o S ny M BDH MP Flow Rate 14 p min 1100 0 100 0 an No er 8 d b 00 09 lv io Min O Sec KE if 00 0 I ene shes Set Core Size Core Size 10 um Threshold C Set Threshold oe a t Volume of This Plot lt a Mean FSC H MeanSSC H CV ae gn Oj 100 00 100 00 0 00 0 00 0 00 o 100 00 100 00 0 00 0 00 0 00 Volume pL of This Plot of All MeanFSC H CVFSC H Median FSC H delt EE o 100 00 100 00 0 00 0 00 mea ee eee 526 2 400 00 100 00 0 00 0 00 Last Run Cumulative Delete Events 7 showwaming _M1 526 216 0 595 961 0 QO 100 00 100 00 _ 0 00 0 00 Events All 1 oe 7 Tom CoE Piot 3 A05 8pk Verification 10mrun Count Volume pl of This Plot of All Mean FL1 H CVFL1 H Median FL1 H a E C Outside R1 Gated on RI Vv s E RRE z This Plot ol 100 00 100 00 0 00 0 00 ae Data Capacity Used ARE D1 ade Dll al art ae An A ae 2 of 24 000 000 Events Figure 4 11 BD CSampler Software Workspace with Empty Sample Well and Empty Plots Click on the RUN or ADD TO button to start a sample collection BD CSampler Software displays and updates d
10. Science is hard Flow cytometry should be easy BD CSampler Software User Guide TABLE OF CONTENTS 1 INTRODUCTION TO BD CSAMPLER mnrannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnrnnnnnnennnnnsnnnnnnennnnnnennnnnsennnn 1 1 1 Installing the BD CSampler rrrannrnnnnnnnnnnennnnnnrnnnnnnnnenrnnnnnnnnennnnsennnnennansnnnnennansennnnen 1 2 BO GSAMPLER SOFTWARE OVERVIEW saved 6 2 1 Starting BD CSampler Softwar e cccccccccccccssececseeeeeeeeeseeeeseeeeseeeeseeeesseeeseeeesseeeess 6 2 2 BD CSampler Software Workspace ccccccsecceceeceseeeeeseeeeteeeeeseeeeseeessaeeeseeeesseeeess 7 2 3 Opening a New BD CSampler Software WorkSpace cccsecceseeeeeeeeeeeeeeeseeeeseeeeess 8 2 4 Loading anad Ejecting a Plate enken dncias eaten aai iaai 8 2 5 Aligning the BD CSampler after a Collision arrrranrrnnnnrrrnnrrrnnnrnranrrrnnnennnnennnnnennnnen 9 2 6 Exiting BD CSampler Software ccccsccccsececceeesceececceeesceeeecaeeescueeeceeeesceeesoeeeess 9 2 Using the Example BD CSampler Software File ccccccccseeeeeeeeeeeeeeeeeeeesaeeeesees 10 3 VALIDATING THE PERFORMANCE OF THE BD ACCURI C6 FLOW CYTOMETER 11 3 1 Running Validation Beads Daily Start Up 2 0 0 0 cecceseeeeeeeeeeeeeeesaeeeeseneeseeeesees 11 3 2 Saving Validation Bead Data ee 15 3 3 Analyzing and Recording Validation Bead Data rrrrnnrrnnnrrnnnrennvrnnnrrnnvrennvrnnnrennven 15 3 4 Monitoring Validation Bead Data
11. decade Range 0 16 77 215 AxIS CD45 kV C linear Min value Max value log 1 16 777 215 GT decade Range O 16 777 215 Apply OK Cancel Figure 4 51 Set Plot Specs Dialog Box with Hide 1st decade Disabled e Click on the OK button to apply the settings and close the dialog box e Open the Compensation Settings dialog box and reset the appropriate compensation values to zero In this example that would be Correct FL2 by subtracting a percentage of FL1 e Perform the fluorescence compensation procedure described in section 4 14 2 The number of events displayed in Q1 LR agrees with the percent value of 18 4 as shown on the plots and Statistics Table in Figure 4 52 Plot 44 HPB C Chex 2257 XI GATE P1 in all s CO4 PE FL2 A la COS FITC FLI A PLOT a tae see L Gated on P1 in all Plot 11 HPB C Chex 2257 Volume pl of This Plot Figure 4 52 Properly Compensated Data After First Decade Is Rehidden 7820099 01 Rev 0 59 BD CSampler Software User Guide 4 15 4 16 4 17 60 Changing Parameters By default BD CSampler Software displays the area parameter signified with a suffix of A but parameters can also be displayed in height width of primary threshold channel only or time To change a parameter e Click on the x or y axis label and select an option from the drop down menu NOTE The time parameter starts counting when the Aun bu
12. 1 Min Figure 5 1 BD CSampler SoftwareAuto Collect Tab The following table describes each of the controls and indicators in the Auto Collect tab 64 7820099 01 Rev 0 BD CSampler Software User Guide Table 5 1 Auto Collect Tab Controls Plate Type Drop down list for setting the plate type Load Plate Eject Plate Moves the sample plate into position to be loaded or ejected from the flow cytometer Plate Name Text box for naming the plate Sample Grid Matrix laid out in the configuration of a multi well plate to help organize experiments and collect data from sample tubes or wells BD CSampler Software acquires each sample into its own well in the Sample Grid The wells are color coded White Does not contain data Colored fill acquisition settings are applied NOTE each unique acquisition setting is indicated by a color change Small blue box Contains data Red outline Currently selected for viewing or collecting data Indicator that displays BD CSampler Software s readiness and system messages Before data collection can begin Traffic Light and Message BD CSampler Software must display a green Traffic Light amp with the message C6 is connected and ready The Traffic Light status is color coded Green BD CSampler Software is ready to collect data or is collecting data Yellow The flow cytometer is preparing to perform an action performing an action other than data collection such as wash or
13. drive to the desktop e Double click on Selectable Lasers Activation Key icon Selectable Lasers Installer exe Figure E 1 Selectable Lasers Installer 7820099 01 Rev 0 113 BD CSampler Software User Guide 114 In the installation wizard choose the directory in which to install the Activation Key The location depends on where BD Accuri C6 Software or BD CSampler Software has been installed In most cases the location will be in one of the following places e C Program Files BD Accuri BD Accuri C6Software ActivationKeys e C Program Files BD CSampler ActivationKeys NOTE If BD Accuri C6 Software and BD CSampler Software are installed on the same computer install the Selectable Lasers Activation Key twice once in the BD Accuri C6 Software directory and again in the BD CSampler Software directory Click on the browse button L to navigate to the correct location Fa Selectable Lasers Setup Choose Activation Keys Directory To install choose the ActivationKeys folder inside your application For example C Program Files BD Accuri BD Accuri C6 Software VActivationKeys C Program Files BD Accuri BD CSampler VActivationKeys Install Cancel Figure E 2 Install Wizard Choose Activation Keys Directory 7820099 01 Rev 0 BD CSampler Software User Guide e Click on the Install button Choose Activation Keys Directory To install choose the ActivationKeys folder inside your application For exam
14. peaks for both FL3 and FL4 DO 6 peak DO 6 peak Gate RT in alli Gate RT in all s 1 000 Count Count sl t o ot o od a a a a a ah Oa FL3 H FLAH AQ 20100308 2633 6 pk 2br AQ 20100308 2633 6 pk 2b2r Gate R1 in alli Gate R1 in all 2 000 2 000 1 500 1 500 Count 1 000 Count 1 000 400 00 oa EE EE JE SE al at at G G t FL3 H FL4H Figure E 9 Evaluation of 6 Peak Validation Beads for the 2 Blue 2 Red Configuration 7820099 01 Rev 0 117 BD CSampler Software User Guide e Confirm proper operation of the 4 blue configuration by comparing the 8 peak distributions for FL3 and FL4 to those obtained with the standard 3 blue 1 red configuration Results for the 4 blue selection should show similar peak profiles for FL3 and FL4 ADT 8 peak 3b 1r A01 8 peak 3b fr Gate R1 in alli Gate R1 in all i Boo 400 3 blue 1 red g E Sa a a al a al ma al at aed a ae afl ag 2 FLS H FLAH AOS 8 peak 4 blue AOS 6 pk 4b std filters Gate R1 in all Gate R1 in all and R3 in alh 4 blue 1 000 Count Count 400 al at at aha an ag gl ad ae ade a a ga FL3 H FLAH Figure E 10 Evaluation of 8 Peak Validation Beads for the 4 Blue Configuration E 3 Annotating Selected Laser Configuration The selectable lasers radio buttons show the most recently selected configuration only BD CSampler Software does not retain this setting for each well Therefore it is
15. recommended to annotate the data wells in the naming field of BD CSampler Software to indicate the laser configuration used during data collection of each well especially when using alternate configurations For example name a sample HPB 4b to indicate that the 4 blue option was selected during data collection 118 7820099 01 Rev 0 BD CSampler Software User Guide A01 HPB 4b LGG EEG TOER 22 at az A3 Ad AB AG A7 AB AQ A10 A11 A12 B1 B2 BS B4 BS BG B7 BS BS B10 B11 B12 C1 C25C3 C4 C55 C6 C7 C8 C91C101C111C12 D1 D2 D3 D4 DS D6 D DS DS B10 011 b12 E1 E25 E3 E49 ES EGE E7 ES ES FE108E118E12 FAR FZEFSREFSAR FS SFG BE JF FS FS EF10 F11 F12 G1 62 63 64 G5 G6 G7 68 G9 610 611 G12 A p C D E F G H H1 H28 HS H4 HS H6 H H H9S H10 H11 H12 3 blue 1 red C6 is connected and ready 2 blue 2 red Figure E 11 Renamed Data Well to Indicate 4 Blue Configuration Laser configurations are not saved in the BD CSampler Software data file or with a BD CSampler Software template Any previously saved BD CSampler Software file will default back to the 3 blue 7 red option when it is opened The last laser configuration used to collect a given well of data is written in the FCS file header using the custom keyword LASERCONFIGURATION FCS file headers can be viewed by opening an exported FCS file in a text editor such as Microsoft Notepad E 4 Optical Filter Placement Due to the unique
16. 1 Table 1 1 BD CSampler Shipping Contents rrrnrrrranrrnnnrerranrrnnnnerrnnrrnnnnennnnrnrnnnennnnennnsnennnnen 1 Figure 1 2 Installing the BD CSampler SIP Collar rrrrarrrnnnnrnnnnnnrnnnnnnnnennnnrnnnnnennnnnnnnnnennnnsnnnn 2 Figure 1 3 Location of Mounting Bolt Holes arrrnrennrennrvnnnernnrvnnrnnnnnnnrnnnrnnnennnennnennsennnnnnnennnen 2 Figure 1 4 Insert BD CSampler Tab into Flow Cytometer cccccseeeeseeeeeeeeeeseeeeseeeeeseeenees 3 Figure 1 5 Secure the BD CSampler to the Flow Cytometet cccccceececseeeeseeeeseeeesaeeeesees 3 Figure 1 6 Secure the BD CSampler to the Flow Cytometet cccccceeceeeeeeseeeeseeeeeeeeeeees 4 Figure 1 7 Connect the BD CSampler Cable to the Flow Cytometer ccsseeeeeeeeeeeeeeees 5 Figure 1 8 BD CSampler with Mat Installed rrronrrrrnnrnrnnnrnnanrnrnnnennnnernnnnnnnnnennnnnnnnnnennnnennnn 5 Figure 2 1 New BD CSampler Software WorksSpace cccsccceceeeeceeeeeseeeeeseeeeseeeeseeesseeesees 7 Figure 2 2 BD CSampler Software Manual Collect Tab Workspace ranrrennnnrvnnnnvrennnnrrennnnn 8 Figure 2 2 Collision Detected WINKOW ccccecccceececceececeeeeeaeeeeseeeesseeeeseueesseeessaeeeseeesaaeeesees 9 Table 2 1 Experimental Design for HPB 4 Color Tutorial File rrarrrnnrrrnrrrrnnennnrrnnnrrnnnennnrnnnre 10 Figur 3 1 Open Bead Tompldle EE 12 Table 3 1 24 Tube Rack Setup for Validation
17. 1 600 000 a Oar ZAR ey cede AM IT ge es a Last Run Cumulative C V st o Events o 0 00 0 Time 0000 Al 0 Microliters o C Outside oOo 0 Events Sec o 0 Events pL o Data Capacity Used 0 of 96 000 000 Events Figure 4 2 New BD CSampler Software Workspace 7820099 01 Rev 0 25 BD CSampler Software User Guide The following sections describe how to set up a sample acquisition and use plots in the Manual Collect tab To collect sample data e Inthe Manual Collect tab do one of the following Select a plate type and name the plate optional Open a template Create new plots optional see section 4 4 Define acquisition settings see sections 4 2 1 and 4 2 2 If necessary acquire some events to define regions gates and other settings Perform analysis in the Manual Collect or Analyze tab 4 2 1 Setting the Fluidics Rate The system can accommodate an upper limit of 10 000 events per second but it is recommended to acquire samples at a rate of 2 500 events per second or less to ensure the best data resolution To set the fluidics rate e Click on the Slow Medium or Fast radio button in the Fluidics section of the Manual Collect tab NOTE It is recommended to start data collection on slow and observe the data rate The setting can then be adjusted to medium or fast if necessary If the data rate is near or above 10 000 total events per second on the Slow setting there are severa
18. 2 9 Adding New Sample Data to a BD CSampler Software File Additional sample data can be added to a BD CSampler Software file that already contains data either by moving to an empty data well before acquisition or by adding to a well which already contains data To add data to a BD CSampler Software file Gently resuspend the sample and load the plate or tube rack NOTE Generally there is no need to perform a backflush between samples Click on a data well in the sample grid If an empty well is selected any plots and gates created previously are still displayed but they do not contain any data as shown in the following figure BD CSampler BD Manual Collect Plate Type 24 tube rack v G Plot 1 ADS Spk Verification 10m run Cj Plot 2 ADS Spk Verification 10m run a GA i i TE No Gating a GATE No Gating Plate Name A05 8pk Verification 10m run L 2 3 4 5 600009 20 33 12 Da aioa aa aa as a Ay Ae AG Rio Ai R a of 4 JG KER ISG BE HET ST c 100 0 1 SETTET the z I I I 1 c1 c2 ca ca c5 ce GF ce ca cao C11 612 ne D1 D2 D3 D4 DS pa D Da DS DIO DiI DIZ S 100 0 E1 E2 E3 E4 E5 EG E7 E8 E9 E10 E11 E12 F1 F2 F3 F4 F5 FO F7 FB FO FAO F11 F12 G1 62 63 64 G5 G6 G7 68 68 G10 611 612 s r EN H1 HZ H3 H4 HS He H7 HB HO H10 H11 H12 a 16 562 550 000 600 000 673 492 FSC H T Ge m De S i Plot 4 A05 Spk Verification 10m run xX GATE R1 53 10 000 12 800 Run Settings Fluidics
19. 20 Figure 4 1 BD CSampler Software Manual Collect Tab rarrrrnnnrnnnnnnrnnnrnnnnrnrnnnnnnnnennnnnennnnen 23 Table 4 1 Manual Collect Tab ControlS arrrnnanrnnnnnnnnnnnnrnnnnnnnnenrnnrnnnnnennnnnnnnnnennnsnnnnennnsnnnnne 23 Figure 4 2 New BD CSampler Software WorkSpace cccccsecccseeeeceeeesaeeeseeeeseaeeeseeeeseeeeeas 25 Table 4 2 Suggested Starting FSC H Threshold Settings for Various Cell Types 0 27 Figure 4 3 Threshold Settings Warning Message rrrrrnrrvvnnnvrenrnnrrernnerennnnerennnvreennnerensnnerennne 27 Figure 4 4 Primary Threshold Drop Down LisSt rarornnrnnnrnnnrnnnrnnnennnennnrnnnrnnnrnnnennnennnenneennsennee 28 Figure 4 5 Threshold Settings Dialog BOX rrrarrrnnnrenranrrnnnnrnrnnrrrnnnennnnennnnsennnnennnnrennnnennnsennnnene 28 Figure 4 6 Plate Type Drop Down Menu rrnanrrnnnrnnnnvnnennnnvnnennnrnnnennnennnrnnnennnennnennnennnennnennsennee 29 Figure 4 7 Plate Name Fv 30 Figure 4 8 Sample Name Field rrrrarrrnnnnrnrnnnrnnnnrnnnnrnnnnnennanennnnnennnnennnnnnnnnnennansnnnnnennnnsnnnnnen 30 Figure 4 9 Run Limits Controls ccesccccctcncsicesauccceststpsntecadedsosdateancedshseccsdstedeuseskedeaptacedededotedcestsant 31 Figure 4 10 BD CSampler Software Workspace after Collecting Samples ccceeeeeeee 32 Figure 4 11 BD CSampler Software Workspace with Empty Sample Well and Empty Plots 33 Table 4 3 Plate Setup for Ending a Data Collection SESSION
20. 24 tube rack assignment Displays rows A D columns 1 6 5 4 Naming the Plate The sample plate can be named to distinguish it from other sample plates in similar experiments optional To name the plate e Name the plate by typing a label in the Plate Name field Auto Collect Plate Type 96 deep well plate v Plate Name Select All Ctrl click to view sample settings Deselect All A10 A11 A12 B10 B11 B12 C10 C11 C12 D10 D11 012 E10 E11 E12 F10 F11 F12 610 611 G12 H10 H11 H12 Figure 5 3 Plate Name Field 7820099 01 Rev 0 67 BD CSampler Software User Guide 5 9 68 Using Sample Sets A sample set is a group of samples that use the same data acquisition criteria such as number of events or threshold setting Acquisition criteria include e Run limits number of total or gated events sample volume or time e Fluidics settings e Wash settings e Threshold s default is FSC H 80 000 Each sample set is highlighted in a different color Twelve colors are available but up to 96 sets can be created colors are reused Figure 5 4 The Auto Collect Tab Displaying Two Data Sets CAUTION When a plate run is started data will be collected in every well with settings applied regardless of whether the well contains previously acquired data 5 5 1 Creating Sample Sets e Select one or more wells to include in the set by doing one of the following e Click on individual wells e Click on the colu
21. 3 Removing VirtualGain To permanently remove VirtualGain from every parameter in every sample in the BD CSampler Software file e Select Display gt Remove All VirtualGain CAUTION This is a permanent action Undo cannot be applied 7820099 01 Rev 0 101 BD CSampler Software User Guide APPENDIX A BD CSAMPLER SOFTWARE MENU 102 QUICK REFERENCE The following table provides a description of all menu options in BD CSampler SoftwareTable A 1 BD CSampler Software Menu Options Open BD CSampler Software Opens a previously saved BD CSampler File or Template Software file or BD CSampler Software template Only one BD CSampler Software workspace can be open at a time New BD CSampler Software Opens a new blank BD CSampler Software File workspace Replaces any previously open workspace Save Saves the open BD CSampler Software workspace under the current name If the file has not already been named BD CSampler Software prompts the user to name the file File as workspace under a new name Save BD CSampler Software Creates a template from the currently open Template as BD CSampler Software workspace All markers regions gates parameter names and sample names are saved without any data points Auto save Settings Allows auto save feature to be enabled or disabled Import FCS File Imports an FCS file previously exported from another BD CSampler Software file to the currently open workspace Only FCS files create
22. 4 Signing In and Signing Out Once user tracking is set up users must sign in for each session 7820099 01 Rev 0 109 BD CSampler Software User Guide To sign in to BD CSampler Software Open BD CSampler Software When prompted enter Username in the text box Sign In Username Password OK Cancel Figure C 7 Username and Password Dialog Box Enter Password in the text box Click on the OK button Use BD CSampler Software as usual BD CSampler Software will continue to log time until the user signs out To sign out of BD CSampler Software Select File gt Quit C 5 Restoring a Forgotten Administrator Password The password file is encrypted and contains all username and password information created in the User Logging feature If the Administrator password is forgotten the password file can be deleted and the Administrator account recreated CAUTION This procedure deletes a user names and passwords each account must be manually recreated To restore the administrator password 170 lf BD CSampler Software is running shut down BD CSampler Software Navigate to the CytometerSupportFiles folder typically in the root directory of the BD CSampler Software computer Locate the Password file and delete the file Follow the procedures in sections C 2 and C 3 1 7820099 01 Rev 0 BD CSampler Software User Guide C 6 Monitoring User Activity Each time a user signs into or signs out of BD CSam
23. 7 8 9 0 11 22 av VA VA R 8 e2 eo pa Bs oo Fe fo Bi Bit Si C1 4 C24 C3 9 C49 C55 C6 c7 ce ca C10 C11 C12 D1 D24 DS D4 DS DE D7 D8 DA D10 D11 D12 EI E2 E3 E4 eS ES e7 es summer FA F2 F3 FA PS FS FTO F8 FO FIG FH FI et 2 og og os os or os og oio ot oi MITTE Figure 4 7 Plate Name Field m Oo on 10 0 Dm 9 4 2 5 Naming the Sample Samples can be named at any time If no text is entered in the naming field BD CSampler Software names the sample according to the well location for example A01 To name the sample e Type the sample name into the text box above the 96 well grid 10 11 12 A10 Add A12 B10 611 Biz Cig 011 C12 DIO D11 Diz E10 E11 E1z Fig Fii Fiz G10 611612 A B C D E F G H H10 H11 H12 Figure 4 8 Sample Name Field 30 7820099 01 Rev 0 BD CSampler Software User Guide 4 2 6 Setting a Run Limit The run limit defines when data acquisition will stop The following parameters can be used individually or in combination to set a run limit e Time e Volume e Number of events in a specified gate If multiple run limits are set data collection stops on whichever limit is reached first Run Settings f Run Unlimited f Run with Limits s 200000 events in Ungated Sample W ls Min o Sec lo uL Figure 4 9 Run Limits Controls To collect samples without setting a run limit e Enable the Run Unlimited check box This requires a manual stop To stop the run a
24. CD45 SSC Gate CD45 SSC Gate 117 858 G00 000 400 000 o S O D 8 3 Pr 153 anit wd aol at at t ae t CD45 FITC FL1 CD45 FITC FL1 CD3 CD4 Gate CD3 CD8 Gate at i N O LL o O R 2 jed LO O o N x O a E ane ar ant 1 af ant CD3 PE Cy5 675 25 FL4 CD3 PE Cy5 675 25 FL4 Figure E 14 4 Blue Configuration and the Optional 780 60 Optical Filter at Detector FL3 Example 2 4 Blue FITC PE PE Cy5 PE Texas Red Human Peripheral Blood Subsets Table E 9 Detector and Filter Configuration for Selectable Lasers 4 Blue Example 2 930 30 or 533 30 675 25 PE Cy5 630 30 PE Texas Red 7820099 01 Rev 0 125 BD CSampler Software User Guide 126 The following images show the results for example 2 The color compensation value to correct the spillover of PE FL2 into the PE Texas Red FL4 detector may be in the range of 70 to 90 CD45 SSC Gate 808 427 500 000 SSC A wit a s CD45 FITC FL1 A 7 2 JE CD8 PTRd 630 BP FL4A sol sd s CD3 PC5 675 BP FL3 A i ak aw Bt 72 at R 72 wo CD4 PE FL2 H so sol EEE D EE CD3 PC5 675 BP FL3 H Figure E 15 HPB Stained with CD45 FITC CD4 PE CD8 PE Texas Red and CD3 PE Cy5 7820099 01 Rev 0 BD CSampler Software User Guide APPENDIX F ENHANCED ANALYSIS FEATURES The Enhanced Analysis module includes several advanced functions in BD CSampler Software e Live or dump gating e Plot and re
25. CD8 cell populations These samples were prepared by staining peripheral blood with directly conjugated antibodies followed by red cell lysis according to standard methods The following table describes the experimental design e Tube 1 Background control unstained e Tube 2 White blood cell gating control CD45 e Tube 3 T cell gating control e Tube 4 Experimental sample Table 2 1 Experimental Design for HPB 4 Color Tutorial File Tube 1 Tube 2 Tube 3 Tube 4 This experimental design does not contain all single stained fluorescence controls but only those required for determining color compensation settings to correct fluorescence spillover For details on correcting fluorescence spillover see section 4 14 7820099 01 Rev 0 BD CSampler Software User Guide 3 VALIDATING THE PERFORMANCE OF THE BD ACCURI C6 FLOW CYTOMETER Perform a validation of the system at least once each day of use This ensures that the flow cytometer is working properly before running experimental samples Using the same BD CSampler Software file each day allows identification of trends over time When collecting validation data BD CSampler Software advances to the next empty well in row A B for 8 peak beads or C D for 6 peak beads Include the date in the Sample Naming Field for each day of validation Start a new validation bead file when all data wells are full Reagents required Spherotech 8 Peak Validation Beads PN 65
26. Capacity Used meter that displays the amount of data storage capacity currently used in BD CSampler Software Gated events can be deleted with the Enhanced Analysis Features Plots Pane Area displaying two rows of plot corrals for graphically viewing data on the selected sample Scroll up or down to view additional plots Each plot corral contains buttons for creating histogram density and dot plots For information on creating and using plots see section 4 4 Statistics Table Table below the plots that displays statistical information on individual plots Collecting Sample Data Events can be added to a well containing data When a data well already contains data the RUN button displays ADD TO Run limits may need to be adjusted to accommodate additional data Each data well holds a maximum of 1 million events BD CSampler Software must display a green Traffic Light and the message C6 is connected and ready to collect data The figure below Figure 4 2 shows a new workspace Only a density plot of linear FSC A vs linear SSC A is displayed The plot is already zoomed to show channels 0 to 1 600 000 on FSC A and 0 to 800 000 on SSC A BD CSampler BD Manual Collect Plate Type tify Plate T v i E Plot 1 No Sample GATE No Gating Plate Name 3 t A01 Click Here To Rename Sample ED JMN D 9 DCE pAn mas 280 Mats Msn f de dess MSG IMO KES VEST VAR 0 200 000 400 000 600 000 800 000 Selecta f Plate Type 0 Ea
27. E wg i E i i m 4 T F 7 oO t T o E un m E ut ut l PE Cy7 cells A01 HPB A01 HFB A01 HFB Gate P3 in alli Gate P3 in all Gate P3 in all 10 000 10 000 12 800 4 000 Count 4 000 Partial data displayed Count Partial data displayed Partial data displayed F al at am ata G st EEE an o ol aol at at at a t CD45 PE Cy7 A FLA A FL2 A AQT APB Gate Mo Gating 4 000 4 000 Count 27 000 40000 125064 events displayed ul at ata afl t FLAA Figure 4 44 Fluorescence Spillover in Different Plots 54 7820099 01 Rev 0 BD CSampler Software User Guide 4 14 2 Correcting Fluorescence Spillover Fluorescence spillover can be removed from plots by applying a mathematical algorithm to collected data This process is often called color compensation or fluorescence subtraction Because data collection on the flow cytometer is digital color compensation can be applied or removed before during or after data collection The color compensation algorithm subtracts a user defined percentage of fluorescence signal from every event thereby redistributing data to lower channels on the fluorescence scale and removing the apparent fluorescence spillover When color compensation has been properly applied to a data set the median fluorescence channel value in non primary detectors for any given single stained control sample should be the same as that of an unstained control sam
28. Figure 4 38 Before and After Using ZOOM Tool cccccccseeeceeeeseeeeeeeeceueeseeeeeueeseeeeseeesaeeenaees 49 Figure 4 39 Plot Spec Dialog Box Set Min and Max Channel Values for the X and Y Axes 50 Figure 4 40 Title Bar with File Name swisdeiccnsurnundernsvins L KE si GGAK kusk dabsteapailat 51 Figure 4 41 Auto Save Settings Dialog BOX rrrnnnrrnnnnnrnnnnrnrannnnnnnennnnrnnnnnnnnnnennnnennnnnennnsnnnnnen 51 Figure 4 42 Save BD CSampler Software File rrrrnrrrrnnrrrrnnrrrnnnenrnnrnrnnnennnnrnrnnnennnnennnnnennnnen 52 Figure 4 43 Save BD CSampler SoftwareTemplate rrrrrnrrrnnnnrnnnnrrrnnnrrnnnrnrnnnrnnnnenrnnnnnnnnen 53 Figure 4 44 Fluorescence Spillover in Different Plots rrrarrrrnnrrnnnnrnrnnnernnnrnrannennnnennnnnennnnen 54 Figure 4 45 Corrected Fluorescence Spillover cccccccccssececeeeeseeeeceecesseeeeseeesaaeeeseeeesseeeeas 55 Figure 4 46 Placing a Quadrant Toolsct ktnueei eee eee 56 Figure 4 47 Statistics Table Displaying Median Values cccccccseeeceeeeeeeeeeeeeseeeeseeesaeeenaees 56 Table 4 4 Fluorochrome Spillover per Channel cccccccccsecccceseeeeeeeeseeeeseeesseeeeseneesseeesses 57 Figure 4 50 Plot Displaying OvercOMPeNSaAtiON ccccccsececeeeeceeeeeeeeceueeseesaueeseeeseeeseeeeseees 58 Figure 4 53 Set Plot Drag and Drop Format Dialog BOX rrrrarrrrnnrerrnnrrrnnnrnnnnernnnrennnnennnnnennnnen 61 Figure 4 54 Plots Selected for Printing
29. Gating a u 7 CD46 PerltP A qo far Figure F 7 New Plot Name 128 7820099 01 Rev 0 BD CSampler Software User Guide F 3 Coloring Events in a Region All events within one or more regions can be designated to appear as a specified color within other histogram or dot plots The event coloring will remain and automatically update during data acquisition NOTE Color will not appear in a density plot To color events e Create a region in a histogram dot or density plot e Double click on the region name and click on the small white square to display a color palette The most commonly used colors appear on the top row E Plot 1 B01 RD At1 GATE Ho Gating Figure F 8 Select a Region to Color e Select a color Figure F 9 Select a Color for a Region 7820099 01 Rev 0 129 BD CSampler Software User Guide e Click outside the region to make the change take effect Events that fall within the region in other dot and histogram plots are now colored and displayed in the same color Plot 2 BOY RD At1 KA E Plot 3 BOY RO 4t1 GATE No Gating GATE No Gating 1 000 000 T a on on 1 000 000 2 000 000 3 195 661 2 000 000 4 094 440 FSC A ZAK DO2 EAE Figure F 10 Colored Events with a Region F 4 Creating Publication Quality Images of Plots High resolution publication quality images are stored in eps format which is vector scalable To create a high resolution image of a p
30. Guide 6 ANALYZING SAMPLE DATA The Analyze tab allows data from multiple samples to be viewed simultaneously using the same plots and gating 6 1 80 Use the tab to do the following View several plots and samples in any combination for easy analysis Compare specific samples from the 96 well grid Create new plots hide plots or copy and reuse plots from the Collect tab View different samples with the same plots Create color overlay histograms Print multiple plots Adjust peak position Viewing the Analyze Tab The Analyze tab is organized into two major sections Setup panel Panel on the left side of the window that contains controls for selecting samples and plots Data display Large area on the right side of the window that shows the sample data in plots and in a Statistics Table When the Analyze tab is opened for the first time the workspace is empty Plots can be copied from the Manual Collect tab or created from scratch Gating strategies that were set up in the Manual Collect tab can be applied in Analyze or new gates can be drawn and new gating strategies set up in Analyze 7820099 01 Rev 0 BD CSampler Software User Guide BD CSampler Plate Type 24 tube rack w E Plot 1C A02 SSC Unity Plot 3C A02 SSC Unity Plot 4C A02 SSC Unity GATE No Gating GATE gt No Gating GATE No Gating Plate Name are to name plate 4 A02 SC U FE MN e gt ele OS IO EPP h PG Sn amp
31. Plot 5 401 8 peak 2651 Plot 6 A041 8 peak 2651 GATE R1 in all ee GATE No Gating os GATE R2in all Figure 3 13 8 Peak Validation Bead Data Select the well containing the most recent 6 peak bead data Adjust the pre drawn region R2 in the 6 peak scatter plot FSC H vs SSC H to encompass the main population similar to the procedure for the 8 peak beads This population should look like an exclamation point The R2 region should encompass the entire exclamation point Figure 3 14 7820099 01 Rev 0 BD CSampler Software User Guide Plot 1 403 6 pk beads x Plot 2 A03 6 pk beads x Plot 3 403 6 pk beads x GATE No Gating GATE R1 in all GATE R1 in all Plot 4 A03 6 pk beads Plot 5 403 6 pk beads Plot amp 403 6 pk beads GATE R1 in all GATE No Gating GATE R32 in all 6 000 7800 Count 4 000 2 000 Figure 3 14 6 Peak Validation Bead Data e Verify that the FL4 H plot is gated on region R2 If it is not click on the GATE button and select R2 on all events from the pop up dialog box e Measure the CV of the top brightest far right peak by adjusting the marker in the FL4 H plot so that it is placed tightly around the peak See the plots in Figure 3 14 for an example of acceptable 6 peak bead data Look for the following e One main population of beads on FSC H vs SSC H e Six peaks on FL4 H e If desired record the following information for each parameter in the BD Accuri C6 Cytometer Log availa
32. Plot Specifications rarrrrnnrernanrrrnnnernnnrrrnnnerranenrnnnrnnnnrrnnnnennasennnnsennnnennnn 36 4 6 Changing the Number of Events in a Plot rornnrnnornnnonnnrnnnnennnnnnnnrnnnnennnnnnnnrnnnnennnnn 37 ar AS T a E 39 Aste TEAMENE 39 4 7 2 Applying a Gate to a PlOt rrranrnnnonnnrnranennnnrnnnrnnnnennnnnnnnrnnnnennnrnnnnernnnennsennnee 41 4 7 3 Creating and Applying Nested Gates rrrrnnrrnranrrrnnnenrnnrnrnnnennnnrnnnnnennnnennnn 42 4 8 Moving and Resizing REGIONS ccccseccseecseeeeeeceeeeaeeceueeeeeeaeeeaeeeneeeseeeneeeaeeeneeenaes 45 4 9 Changing the Number of Events in a Plot ronnnnanonnnnennnnnnnnennnrennnennnnennnnnnnnrnnnnennsenn 46 10 EFN CS suiacautosaacaadesiacandasicdantesii canto adacaninatacantoudacanteaiacanteuiataadeatacantemiasanceate 47 4 11 ZIP endda 49 4 11 1 Basic Zidane mene dine dedeed shibaieGanccnindsriaaiedarbanieasnte 49 4 11 2 Zooming to a Specified Channel Range ccccceecccssececeeesaeeeeseeeesaeeeeees 50 4 12 Saving a BD CSampler Software File rrrrnnrrrnnrrrrnnnerrnnrnrnnnennnnrrnnnnennnnennnnnennnnennnn 50 2 PRUIO o WING APCS EEE 51 4122 Manually Saving Files ziscwsscececnnsecacnencinsennsentewsneasesaensestaasesncesbensexteeceenedeenes 52 4 13 Creating a BD CSampler Template rrrarrrrnnrrrnnrrrrnnnerrnnrnrnnnrrnnnrrnnnnennnnennnnnennnnennnn 52 7820099 01 Rev 0 i BD CSampler Software User Guide ii 4 14 Understanding Fluorescence SpillOV
33. agitate or a non critical error has occurred Red A critical error has occurred Run Limits Contains a set of controls for defining criteria for automatically stopping the data collection See section 5 6 3 for details size See section 5 6 1 for details samples See section 5 6 5 for details Set Threshold Sets the event threshold to gate out debris and noise from samples The default value is 80 000 on FSC H See section 5 6 2 for details on setting threshold values Settings sample set See sections 5 5 1 through 5 5 5 for details See section 5 6 4 for details Open Run Display Opens the run display for starting and stopping a run viewing data acquisition counters and viewing two plots of data See section 5 8 for more information Sample Annotation Table Contains fields for naming samples renaming parameters and adding a notation to each sample Information can be entered manually for each sample or copied and pasted from a spreadsheet program 7820099 01 Rev 0 65 BD CSampler Software User Guide 5 2 5 3 66 Running a Sample Plate in Auto Collect The following procedure describes a typical setup and data collection workflow using BD CSampler Software Perform pre acquisition functions in the Manual Collect tab such as creating plot displays and gates Click on the Auto Collect tab Create sample sets with specified acquisition agitation and wash settings NOTE Agitate settings are app
34. aligned by clicking on the corresponding blue well in the pop up sample grid the gray well indicates the standard sample currently selected Ensure that this plot has been zoomed to the required level before setting VirtualGain Standard Sample Lg 3 4 3260 71 9 BG ee Aa AD AA AB PB AT ABT AG AID AIT A12 61 82 63 B4 86 BO 87 OG BO BIO B11 B12 ct C21 C3 04 CS OG I C7 O8 I OG C101C111C12 01 D I 03 D 06 DO D7 06 DG 010 015 012 E11 G2 G3 64 65 66 67 69 EG E10 E11 E12 F FS FO F7 FS FQ FID F11 F12 G1 I 62 I 63 04 G6 66 I G GS OG 161016111012 A B c D E F G H Ht H2 HD G4 HS HOT H OMB MG MIO HIT HZ Figure 9 4 Pick the Sample to Align e Move the peak definition marker in the Sample to Align plot to the center of the peak to align 98 7820099 01 Rev 0 BD CSampler Software User Guide Standard Sample Figure 9 5 Move the Peak Definition Marker Click on the Preview button to view the aligned sample with VirtualGain applied BD CSampler Software aligns the peak of interest in both plots Repeat steps 9 10 to make additional adjustments if needed To align additional samples exactly as the first aligned sample select the This sample and radio button and click on the well s in the pop up sample grid to be included If the other samples need a different amount of VirtualGain set VirtualGain separately for each sample Click on
35. becomes Plot 2C Plot 3 FSC A becomes Plot 3C Note Gating information will not be copied OK Cancel Figure 6 2 Selecting Plots to Copy from the Collect Tab e Click on the OK button to close the dialog box BD CSampler Software adds the selected plots to the Plot List in the Analyze tab 82 7820099 01 Rev 0 BD CSampler Software User Guide Plot List Plot 1C FSC AJ SSC A Plot 2C FL1 4 Plot 3C F5c 4 Set Color Compensation Figure 6 3 Plot List Containing Copied Plots 6 2 2 Creating Plots e Click on an empty plot corral e Click on one of the following icons under the Sample Grid e Histogram ki e Dot e Density e Overlay Histogram see section 6 2 3 for details e Click on the sample well to view data 6 2 38 Creating an Overlay Histogram Creating an overlay histogram allows comparison of multiple distributions from up to 96 different samples at the same time To create an overlay histogram e Click on an empty plot corral e Click on the Overlay Histogram Tool to open a blank single parameter FSC A plot 7820099 01 Rev 0 83 BD CSampler Software User Guide Plot Mo Sample x GATE Mo Gating 000 000 10 000 000 16 777 215 FSC A sree Figure 6 4 Blank Overlay Histogram Plot e Click on the x axis label FSC A and select a different parameter in the drop down list if desired e Click onthe GATE button and apply a gate if desired see section 4 7 2 for
36. box do the following for each axis e Select the desired parameter for display from the drop down list e Select the inear or log radio button to specify how data are scaled All parameters are collected with linear amplifiers on the flow cytometer and therefore all channel values are linear Selecting the log view of a parameter simply means the linear channel values are graphed on a logarithmic scale e Type in the minimum and maximum channels to set the channel range to view Enable or disable the Hide 1st decade check box It is often beneficial to unhide the first decade of a given parameter before applying fluorescence compensation See section 4 14 e Doone of the following e Click on the Apply button to apply the changes without closing the dialog box e Click on the OK button to apply the changes and close the dialog box e Click on the Cancel button to close the dialog box without applying the changes 4 6 Changing the Number of Events in a Plot The number of events displayed in all plots across all samples can be changed in order to improve data visualization or to normalize data sets This option allows visual removal of a number of events from the plot without deleting data Flot 4 403 HFE COS C045 p Plot 4 203 HFE COS C045 GATE No Gating GATE No Gating 700 000 400 000 600 000 800 000 events displayed ca aa oo o F o o o 4 JE267 1 231352 20 1 S00
37. details e Select the samples to be overlaid from the 96 well grid Plot 5 404 Unstained 202 HFE CD4 x GATE No Gating 10 000 12 800 S00 O00 1 185 891 FSC A oes H Figure 6 5 Overlay Histogram Plot with Data 84 7820099 01 Rev 0 BD CSampler Software User Guide e Click on the Overlay Histogram Legend Tool amp to view a legend for the overlay histogram Overlay Histogram Legend x P A05 RBC 1 t1 E B05 CD ch t1 co1 CD Ch BC t1 Figure 6 6 Overlay Histogram Legend To change the color of one or more overlay plots click on the square in the Overlay Histogram Legend and select the desired color from the pop up color palette Plot 2 405 RBC 1 1 605 CD Ch t1 J a GATE No Gating mre Overlay Histogram Legend 405 RBC 1t1 B05 CD Ch ti E coi CD Ch BC ti 1 000 000 2 000 000 3 799 603 FSC 4 EN 7 Figure 6 7 Overlay Histogram Legend with Color Palette 6 3 Viewing Plots To view a plot in the Plot List e Click on an empty plot corral in the Analyze tab e Selecta plot in the Plot List Figure 6 8 BD CSampler Software displays the plot without any sample data 7820099 01 Rev 0 85 BD CSampler Software User Guide NOTE Any gating tools markers regions etc carried over from the Collect tab are renamed for example P1 in Collect is P2 in Analyze When these new markers or regions are adjusted in the Analyze tab the origi
38. e Doone of the following In the text box next to the FL button type an arbitrary percentage of the signal to subtract Use the C Comp Calculator Excel spreadsheet provided by BD Accuri available on the BD CSampler Software CD or flash drive or at www accuricytometers com resources manuals to calculate the subtraction values See the appropriate tab of the C Comp Calculator for your instrument serial number for suggested fluorochrome specific spillover values Table 3 3 is an example Table 4 4 Fluorochrome Spillover per Channel FITC PE PerCP PerCPCy5 5 FL1 530BP N A 3 2 0 00 0 0 PE Cy7 APC FL2 585 BP 6 5 N A 0 00 0 00 FL3 670 LP 1 0 19 5 N A N A FL4 675 BP 0 0 0 0 3 00 12 00 7820099 01 Rev 0 57 BD CSampler Software User Guide e Click on the Preview button to update the plots and Statistics Table e Inthe Statistics Table observe the values in the Median column of the FL channel of interest e Repeat steps 5 9 until the median value for the UL or LR quadrant is equal or nearly equal to the median value of the negative population LL This value is called the compensation value The figure below shows the median values highlighted in blue of This Plot of All MeanFL1 A Mean CD45 PE Cy7 A CVFL1 A CV CD45 PE Cy7 A MedianFL1 A Median CD45 PE Cy7 A 1 145 5 Figure 4 49 Results of Subtracting Spillover e To apply the fluorescence subtraction to all sam
39. file before the fcs extension BTIM The beginning time of acquisition Unchangeable of the first event PnS PROJ the last event VOL Collecting more events would change the total volume BEGINSTEXT Unchangeable beginning of the text section SENDSTEXT Default FCS 3 0 tag to mark the Unchangeable end of the text section BEGINANALYSIS Default FCS 3 0 tag to mark the Unchangeable beginning of the analysis section ENDANALYSIS Unchangeable end of the analysis section BEGINDATA Unchangeable beginning of the data section SENDDATA Default FCS 3 0 tag to mark the Unchangeable end of the data section 134 7820099 01 Rev 0 BD CSampler Software User Guide CUSTOM TAGS IN FCS FILES EXPORTED BY BD CSAMPLER SOFTWARE Table G 2 Custom FCS Tags parameter N It is always 7 224719870049579 BDACCURI4COLORCOMP A list of the percent values as Determined by values entered entered into the color into Color Compensation compensation window dialog BDACCURICAPTUREDDATE The date of the last time the Defined by the beginning time represented sample was of the most recent acquisition acquired into expressed in Set by the computer s clock milliseconds since Jan 1 1970 PNVIRTUALGAIN The virtual gain set for Set by user in the VirtualGain parameter N where 1 0 window means no virtual gain SAMPLE Value is either the well code Can be changed by renaming or the sample rename if one
40. optical layout of the BD C6 flow cytometer it is critical that any optional filters used with the Selectable Lasers Module are placed in the proper position for optimal performance WARNING The 630 30 bandpass filter provided with the Selectable Lasers module should only be used when operating in the 4 blue configuration Using the 630 30 filter when operating in any other configuration may damage the corresponding detector due to unfiltered red laser signal Use the tables below as a guide to optical filter placement for various fluorochrome combinations Table E 1 3 Blue 1 Red Configuration 1 Standard Filters FL1 530 30 FITC GFP CFSE or 533 30 FL2 585 40 PE PI PE Texas Red FL3 670 LP PerCP Cy 5 5 PE Cy5 PE Cy7 7820099 01 Rev 0 119 BD CSampler Software User Guide 120 FL4 675 25 APC Alexa 647 PE Cy5 Table E 2 3 Blue 1 Red Configuration 2 530 30 FITC GFP CFSE or 533 30 FL3 610 20 PI PE Texas Red FL4 675 25 APG Alexa 647 PE Cy5 NOTE When operating in the 3 blue 1 red configuration place either the 610 20 bandpass or the 670 LP filters in position FL3 For best results when analyzing PE and PE Texas Red PE TR simultaneously select the filters with the following signal intensity considerations in mind e PE bright PE TR moderate to bright FL3 670 LP e PE dim to moderate PE TR any level FL3 610 20 e PE bright PE TR dim may be difficult to separate consider using the 4
41. package instructions according to package instructions Select well A1 in BD CSampler Software Plate Type 24 tube rack il Plate Name Click here to name plate AO1 8 Peak Beads I 2 3 4 5 6 7 8 9 10 11 12 fa naa naa a in a R E B1 Br B3 BJ BS BG B7 BB B9 B10 B11 B42 Ci cz cs C4 4 cC a ES El Be en ee D1 D2 DS D4 p DG D7 D8 DA D40 D11 DAZ SBS BSB amp amp amp E19 E11 en MELET 1 2 co es oe o7 ce ce oooi or MITT Figure 3 2 Select Well A1 I nm mM Oo 54 073 Enable the Run with Limits radio button in the Instrument Control Panel Run Unlimited Run with Limits fs0000 events in ungated Sample lw 15 Min jo Sec E fo ul Figure 3 3 Run Limits Disable Run Unlimited Enable the Time check box next to the Min and Sec fields in the Instrument Control Panel and type in a run time of fifteen minutes Select the Fast radio button in the Fluidics section of the Control Panel Click on the RUN button to rinse the SIP When prompted save the file 7820099 01 Rev 0 BD CSampler Software User Guide Once the run is finished click on the Delete Sample Data button to delete data collected during the rinse Run 8 Peak Validation Beads Select the first empty data well in rows A B This should correspond to the tube containing 8 peak beads Disable the Time check box next to the Min and Sec fields enable the Events check box and type 50 000 into the Events field e Select Ungate
42. statistics can be exported in two ways either as a PowerPoint file or as an Excel spreadsheet To export data Do one of the following in the lower right hand corner of the Batch Analysis tab e Click on the Export to PowerPoint button e Click on the Export Stats to Excel button Figure F 12 Export Buttons in the Batch Analysis Tab In the Save dialog box navigate to the destination folder and type a file name BD CSampler Software exports the file to the specified location 7820099 01 Rev 0 BD CSampler Software User Guide APPENDIX G FCS KEYWORDS This appendix lists all of the keywords BD CSampler Software uses in FCS files STANDARD FCS TAGS IN FCS FILES EXPORTED BY BD CSAMPLER SOFTWARE Table G 1 Standard FCS Tags extension Well Grid SDATATYPE The data type of the actual Unchangeable values for each event It is always I for unsigned binary integers SMODE The mode of the data It is Unchangeable always L for list mode where the data is in the order described by the Pn keywords written least to most significant It is always 4 3 2 1 SNEXTDATA The byte offset for an additional Unchangeable dataset in the file BD CSampler Software files always specify 0 since the files only contain 1 dataset SPAR Total number of parameters Unchangeable stored in the dataset All datasets have 14 parameters PnB For parameter N the number of Unchangeable bits for each binary value The number is
43. the Apply button to apply VirtualGain to the data BD CSampler Software displays a black asterisk under the Sample to Align plot to indicate that VirtualGain has been applied to the specified parameter for that sample 7820099 01 Rev 0 99 BD CSampler Software User Guide 9 2 100 Standard Sample gt o 2 P 2 g v amp Y Z 19 Figure 9 6 Black Asterisk Identifier e Click on the Close button to close the VirtualGain dialog box Viewing VirtualGain When VirtualGain is applied to a sample BD CSampler Software displays a black asterisk under the parameter label in the associated plot The asterisk is color coded e Black VirtualGain has been applied e Gray BD CSampler Software is currently displaying the original data Plot 11 Sample 23 GATE Pe Figure 9 7 Plot with Black Asterisk Overlays automatically display VirtualGain when it is applied Figure 9 8 The asterisk is not displayed in overlays when VirtualGain is applied to some or all of the samples in the overlay 7820099 01 Rev 0 BD CSampler Software User Guide Plot 12 Sample 91 03 02 _ x GATE Fe Figure 9 8 VirtualGain Applied in an Overlay Histogram To toggle between views with VirtualGain applied and not applied e Click on the asterisk in the plot Plot 11 Sample 23 x Plot 11 Sample 23 x GATE Fe GATE F2 Figure 9 9 Toggle between VirtualGain Applied Left and Not Applied Right 9
44. the sample in the application exists If no rename exists importing an FCS file into a different well than the one collected in will change this value more data analysis and data sections of the FCS file FCS 3 0 DEFINED TAGS NOT IN BD CSAMPLER SOFTWARE EXPORTED FCS FILES Table G 3 FCS Tags not in BD CSampler Software Exported Files COMP Amount of fluorescence compensation employed during collection This replaces the DFCiTOj tag from FCS 2 0 TIMESTEP Absolute measure of time used in kinetic analysis UNICODE Enables usage of certain keywords in non English languages This is optional PnE For parameter N this denotes if linear or logarithmic amplifiers are used BD CSampler Software always uses 0 0 because BD CSampler Software always saves linear values for the data Mandatory in 3 0 When time is collected the keyword value of the time parameter name must now be the string TIME An optional 16 bit Cyclic Redundancy Check has been added to the end of each dataset 7820099 01 Rev 0 135 BD CSampler Software User Guide APPENDIX H EXAMPLE CSV FILE The following table is an exported bead log file in csv format Each row represents the parameter data associated with a single event The number of rows will equal the total number of events in the data file A data well that contains 125 000 events also contains 125 000 rows of data in the spreadsheet display of the csv data The Time value is
45. the wash station after 15 minutes in the idle position 3 2 Saving Validation Bead Data By default BD CSampler Software automatically saves data at the end of each sample run Data can also be saved manually at any time For information on saving data see section 4 12 3 3 Analyzing and Recording Validation Bead Data After the bead data is collected analyze the data using the Manual Collect tab of BD CSampler Software to ensure that the flow cytometer is functioning properly e Click on the well that contains the most recent 8 peak bead data rows A and B 7820099 01 Rev 0 15 BD CSampler Software User Guide 16 On the first FSC H vs SSC H plot scatter plot in the bead file adjust the pre drawn region R1 to encompass the main population by dragging the border of the region see Figure 3 10 R1 should contain at least 80 of total events NOTE There is usually a shadow population called bead doublets or clumps that is slightly higher in FSC H than the main cluster of beads this is normal for these beads Do not include the shadow group in R1 Mot 1 E01 amp Peak Beads GATE No Gating Bead doublets Figure 3 10 Plot with Bead Doublets Verify that the next three plots FL1 H FL2 H and FL3 H are gated on scatter region R1 and that the plots display the message R1 in all next to the GATE button Figure 3 11 If it is not displayed click on the GATE button and select R1 on all events from t
46. tubes any type of plastic Other tubes are not supported e Use sample volumes between 300 uL and 2 mL Never acquire more than 750 uL from a single tube on Medium fluidics setting or 1500 uL on Fast fluidics setting Adjust sample concentrations to fall approximately within the range of 1x10 and 5 x 10 cells or particles per mL e Acquire data on Medium or Fast fluidics settings only Custom fluidics settings above 15 uL and 16 um may be used but must be validated by an independent control count bead e Only acquire once from any sample tube Sample height within the tube is critical Replicate measurements must be obtained from separate sample tubes made by aliquoting sample into the appropriate number of equal volumes e Always compare the same stop limit types Do not compare concentrations collected with volume stop counts to event stop counts e Always validate accurate counting by using a reference count bead in the experimental buffer using the same sample volume and tube as in the experiment If bead counts are within 20 of the expected value based on information provided by the bead manufacturer proceed with sample collection If bead counts are not within 20 of expected values proceed with fluidics calibration as described below e Perform the following in order 1 Ensure that the fluid levels in the Sheath Cleaner and Decontamination bottles are sufficient to cover the inlet tubing and that there are no
47. well corresponding to the second tube of water e Set the time limit for two minutes and the fluidics speed to fast e Click on the RUN button e When the run is finished eject the plate tube rack 7820099 01 Rev 0 BD CSampler Software User Guide 4 4 Creating Plots Three plot types are available for viewing data histogram density and dot plots a o oo a o oo a o m o I Flot 1 201 Unstained x GATE Ho Gating 00 000 1 000 000 1 600 000 JEF SYKE Figure 4 12 View of a Plot Each plot contains a set of gating and marker tools and a set of viewing tools e Gating and marker tools Gate button at Opens the Change Gating dialog box for applying gates to a plot Polygonal Gating Tool 9 For drawing irregularly shaped gates around a population of events Rectilinear Gating Tool 2 For drawing a rectilinear gate around a population of events Quadrant Gating Tool 4 For gating the plot in quadrants Vertical Marker Tool For gating histograms to the right or left of a vertical marker Horizontal Marker Tool For gating histograms within a horizontal marker e Viewing tools Plot Spec Tool Opens the Set Plot Specs dialog box for changing the x and y axis parameters scaling the plot and setting log or linear view Zoom Tool amp Defines the Zoom range Expand Tool amp Undoes one Zoom level To create a new plot e C
48. wizard choose the directory in which to install the Activation Key The location depends on where BD CSampler Software has been installed In most cases the location is in C Program Files BD Accuri BD CSampler ActivationKeys If necessary use the browse button to navigate to the correct folder pg User Tracking Setup e Choose Activation Keys Directory To install choose the ActivationKeys folder inside your application For example C Program Files BD Accuri BD Accuri C6 Software VActivationKeys C Program Files BD Accuri BD CSampler ActivationKeys C Program Files BD Accuri BD CSampler Software ActivationKey Fond Install Cancel Figure C 2 Installation Dialog Box Click on the Install button BD CSampler Software displays a confirmation message after successful installation 7820099 01 Rev 0 107 BD CSampler Software User Guide C 2 Using the Tracking Feature for the First Time To use the tracking feature for the first time Open BD CSampler Software When prompted type admin in the Username text box Sign In EE Username Password OK Cancel Figure C 3 Username and Password Dialog Box Type Admin in the Password text box case sensitive The Administrator password can be changed later Click on the OK button Use BD CSampler Software as usual C 3 Adding Deleting and Modifying User Accounts The administrator can add new u
49. 0 C11 C12 D7 DS D9 D10 D11 D12 E7 E8 E9 E10 E11 E12 FF F8 FQ F10 F11 F12 G7 G8 69 G10 G11 612 H7 HS H9 H410 H11 H12 Make a new plot hal 6 bal a 50 000 100 000 50 000 100 000 Analyze Plot 2 A02 HPB CD45 PE Cy 7 xX 5 Plot 3 A04 HPB CD3 CD4 CD45 C E Plot 4 A04 HPB CD3 CD4 CD45 C GATE gt No Gating o GATE P1 sne GATE Q1 UL in P1 gas 158 177 wt D b CD45 PE CY7 A wo CDS APC A Prd wl at a 21 952 200 00 400 000 537 813 FSC A moe SUK Plot 2 402 HPB CD45 PE Cy 7 Plot 3 402 HPB CD45 PE Cy 7 E Plot 4 A02 HPB CD45 PE Cy 7 GATE No Gating I GATE P41 r GATE Q1 ULin P1 158 177 ut CD45 PE Cy7 A ud wl a a 21 952 200 000 400 000 537 813 FSC A oor AR Figure 6 9 Analyze Tab Two Samples with the Same Plots Compare data and statistics between samples 86 7820099 01 Rev 0 BD CSampler Software User Guide 7 7 1 7820099 01 Rev 0 CREATING A STATISTICS TABLE The Statistics tab provides a way of tabulating data from multiple samples in one master table It also allows users to do the following e View statistics for some or all samples e Display statistics of collected or imported samples e List all plots created on the Manual Collect and Analyze tabs e Display all plot names gates and associated statistics e Copy and paste data into a spreadsheet Viewing the Statistics Tab The Statist
50. 00 0002 000 000 3 000 0003 994 576 ge MD ge ort Gar tag I ENER CD3 APC Cy7 FL3 780 BP CD3 CD4 Cells R2 Gate w t CD45RO PE FL2 A vi ae Tir we ape CD45RA FITC FITC FL1 wt CO45RO PE FL2 A we wi oh wt ww CD45RA FITC Figure E 12 Gating Example Using the 2 Blue 2 Red Configuration 7820099 01 Rev 0 123 BD CSampler Software User Guide Example 2 2 Blue 2 Red with BD Cytometric Cytokine Bead Array CBA 30 Plex Bead Mixture BD 51 9004679 The following images show the results for bead separation using the entire 30 plex bead mixture CO2 CBA red 2b2r C02 CBA red 2b2r og Gate R1 in all ce Gate R1 in all 3 3 R T x ia Ea 2 5 5 3 a ee ee ee ee s sd WB 8 3 780 BP FL3 A 780 BP FL3 H Unzoomed View Area Zoomed View Height sFigure E 13 BD Cytometric CBA 30 Plex Bead Mixture collected on the BD Accuri C6 run in 2 Blue 2 Red mode using the Selectable Lasers Module PN 653126 E 5 2 4 Blue Configuration Examples Example 1 4 Blue FITG PE PE Cy5 PE Cy7 Human Peripheral Blood Subsets Table E 8 Detector and Filter Configuration for Selectable Lasers 4 Blue Example 1 530 30 FITC or 533 30 780 60 PE Cy7 675 25 PE Cy5 124 7820099 01 Rev 0 BD CSampler Software User Guide The following images show CD45 FITC vs SSC gating of lymphocytes zoomed in left zoomed out right HPB was stained with CD45 FITC CD8 PE CD3 PE Cy5 and CD4 PE Cy7
51. 000 FSL A Sample Selector Add rows to your master statistics _table by selecting samples Add to Preview Table Sample Name All Samples T AL Unstained 202 HPB CD45 PE C AOS HPB CO3 CD45 and HPB COS CD4 Figure 7 5 Plot Preview The plot preview is available for viewing only The zoom level and other plot settings must be modified in either the Analyze or Manual Collect tab 7820099 01 Rev 0 89 BD CSampler Software User Guide 7 4 Copying Data into Other Applications Data can be copied and pasted from the Master Statistics Table into most Microsoft Office compatible applications To copy data e Use the mouse to highlight the fields The column and row headers corresponding to the selected data fields are automatically copied e Press Ctrl C to copy the data e Inthe Microsoft application press Ctrl V to paste the data NOTE See Appendix F 4 for instructions on Batch Analysis 90 7820099 01 Rev 0 BD CSampler Software User Guide 8 8 1 8 2 MAINTAINING THE BD ACCURI C6 FLOW CYTOMETER To maintain optimal performance follow the routines in this chapter on a regular basis See the BD Accuri C6 Flow Cytometer Instrument Manual for mechanical maintenance procedures such as replacing tubing Cleaning the SIP Run the Backflush Cycle to clean the SIP and remove clogs at the b
52. 000 1 000 000 1 600 000 S00 000 1 000 000 1 600 000 ape ESKE agpo Bae Figure 4 15 Before and After Changing Events Displayed To change the events displayed in a plot e Select Display gt Events Display Settings 7820099 01 Rev 0 37 BD CSampler Software User Guide 38 Do one of the following in the Events Display Settings dialog box To view all collected events select the Show all events radio button To view the first N events of a sample select the Display first radio button and type a number in the events collected field To view a specified percentage of the whole in a pseudo random manner select the Display radio button and type a percentage to view for example if 20 is selected every fifth event is displayed Events display settings hen displaying plots C Display First 50000 events collected C Display 20 percent of events collected Apply Cancel Figure 4 16 Events Display Settings Dialog Box Do one of the following Click on the Apply button to apply settings without closing the dialog box Click on the OK button to apply settings and close the dialog box Click on the Cancel button to close the dialog box without applying settings BD CSampler Software updates the display and shows a message in the plot that some events are not being displayed Flot 1 404 Unstained x GATE No Gating Events display settings applied Z00 000 400 000 600 000 800 000 events displa
53. 1 C12 DAO D11 biz E10 E11 E12 FIO F11 Fiz G10 G11 Giz B C D E F G H H10 H11 H1i2 Figure 3 6 Select Well for 6 Peak Beads Verify that the check box by Events is still enabled and set at 50000 and that Ungated Sample is still selected from the drop down list Run Settings f Run Unlimited f Run with Limits i 50000 events in Ungated Sample lo Min 0 Sec lo uL Figure 3 7 Run Limits 50000 Events Click on the RUN button NOTE The R2 region may not encompass the main population of bead events on the FSC H vs SSC H plot This is common and acceptable at this stage Name the sample with a name that includes the date processed E01 6 Peak Beads 20090218 Figure 3 8 Sample Name 6 Peak Beads When the collection is finished click on the Wash button to minimize sample carryover End the Procedure 14 Place a tube with 2 mL of filtered deionized water in the A1 position of the 24 tube rack and advance to the data well in BD CSampler Software Select the Time check box Min Sec in the Instrument Control Panel and set it for two minutes 7820099 01 Rev 0 BD CSampler Software User Guide Run Settings f Run Unlimited Run with Limits 50000 events in Ungated Sample 2 Min O0 Sec o HL Figure 3 9 Run Limits 2 Minutes e Click on the RUN button e When the run is finished leave the tube on the SIP The BD CSampler will automatically place the SIP in
54. 2 Ot 02 09 65 05 06 OF DE OG 010 O11 O12 Et 2 O 4 O 6 EF OG EG EO Ett 12 Fa FP UD F FS FH FY FG FO FIO Fett FI 01 G2 OG 66 65 G6 GF OG OG 610 OTt 612 Mt MZ OG Me HA H OM OM OMG MIO TT Hi 3 bive I red io i Cb it copruected ard ready C 2odwe2 res C blue amp Stew Medium Fest Fiew Rete 14 pUmie in Usgates Sampie v Cere Size 10 pm bh Min See 3 yi Set Threshold Delete events of Mieemume t0 T Da aoi collect events outside r PM T lems Sen 80000 y lems man Wash Settings App satings Remove Settings Agitate Plate a ls Run Hongaatally e ane X every Weis ane l Rya Vertically f every Mia JOPEN RUN DISPLAY Figure 5 6 Samples in a Set Are the Same Color 5 5 2 Viewing Sample Settings To view data acquisition settings for a particular well e Press Ctrl and click on the well of interest 5 5 38 Modifying Sample Settings To modify a set after it is created e Doone of the following e Double click on a well in the set to select the entire set e Click on one or more wells within the set e Adjust the data acquisition settings 7820099 01 Rev 0 BD CSampler Software User Guide 5 6 e Click on the Apply Settings button The selected wells change to a new color 5 5 4 Saving a Sample Set BD CSampler Software saves all sample settings each time the Apply Settings button is clicked Data is automatically saved during a run after eac
55. 2 3 Creating an Overlay Histogram rrrnnrnnnnrnnnnennnnnnnnrnnnnennnrnnnnennnrennnnnnnnennsennnre 83 6 3 NVIGWING ad 0 NN 85 GREATINGA STATISTICS TABLE av 87 7 1 Viewing the Statistics Tab ccccccccccsececosecsceeessceeecouecenceeenceeseccecescenenseesecteeesceeeesees 87 7 2 Creating the Master Statistics Table rarrrrnnrerrnnrrrnnnerrannnnnnnerrnnrnnnnnennnnrnrnnnennnnennnn 88 7 3 Previewing a Plot in the Statistics Tab rrrrrnrrrrnnrornnnrrranrnnnnnerrnnrnnnnnennanrnvnnnennnnennnn 89 7 4 Copying Data into Other Applications rrrnnrrrrnnrrnnnnerrannrrnnnerrnnrrnnnnennanenrannennnnennnn 90 7820099 01 Rev 0 BD CSampler Software User Guide 8 MAINTAINING THE BD ACCURI C6 FLOW CYTOMETER rrnunnnnnnnnnnnnnnnnnnrnnnnnrnnrnnnnennnnnn 91 SA Cleaning the SIP users an ea a A EE aa 91 B22 Gleaningiine FOwWCOEeN censes Pee 91 821 FROM MING Te UNA Cy Cl aa 91 8 2 2 Running an Extended Clean of the Flow Cell rrrnrrrnrnrrnnnnrrnnnnrnvnrrennnrrenn 92 8 3 Cleaning the Fluidics Lines cccccceeccceeeceececeeeeceeeceuseceeeeceecsusenseessueessueeseeesseeens 92 8 4 Decontaminating the FIuidics rrarennrrnnnrvnnrvnnevnnevnnennnennnrnnnennnennnennnennnrnnnennennsennee 93 8 5 Using the BD Accuri C6 Flow Cytometer for Precise Volume Measurements 93 86 Tema SUD DON HSE 95 9 ADJUSTING PEAK POSITION WITH VIRTUALGAIN 0 cccccceecceesssseeeeeseeeeeeeeseeeeeeeeas
56. 24 146225 189713 1183340 664650 850548 73594 3339490 755878 108595 519183 1555340 567177 670857 90865 FL1 H 486 274 443 501 591 110 AG 200 154 259 432 A27 319 150 798 415 669 34 5642 229 204 631 340 287 Aja J FL2 H 60 114 114 152 96 55 70 79 208 177 AG 56 KRASS 1524 131 66 124 108 205 81 97 K FL3 H 161 438 780 118 586 182 275 431 345 702 274 314 414 823 343 121 668 1869 364 464 330 47 243 187 486 L FL4 H 382 383 652 506 629 298 362 458 2 8 631 594 229 732 AA 454 234 252 293 2551 318 390 613 380 407 378 M Width 82 75 78 86 73 71 fi 75 81 82 76 78 82 79 79 49 76 51 82 57 79 79 74 N Time 137 137 137 137 137 137 137 137 137 137 137 137 137 137 137 137 137 137 137 137 138 138 138 138 138 138 7820099 01 Rev 0 BD CSampler Software User Guide Regional Offices bdbiosciences com offices Asia Pacific Singapore Tel 65 6861 0633 Fax 65 6860 1590 China Tel 86 21 3210 4610 Fax 86 21 5292 5191 India Tel 91 124 2383566 Fax 91 124 2383224 25 26 Canada BD Biosciences Toll Free 888 259 0187 Tel 905 542 8028 Fax 888 229 9918 bdbiosciences com ca canada bd com Australia New Zealand Australia Toll Free 1800 656 100 Tel 61 2 8875 7000 Fax 61 2 8875 7200 bd anz bd com New Zealand Toll Free 088 572 468 Tel 64 9 574 2468
57. 3144 supplied with the initial flow cytometer shipment Spherotech 6 Peak Validation Beads PN 653145 supplied with the initial flow cytometer shipment e Sheath fluid Deionized filtered water 0 2 um filter plus Bacteriostatic Concentration Solution PN 653156 supplied with the initial flow cytometer shipment 3 1 Running Validation Beads Daily Start Up Setup e Verify that the Bead Template has been copied to the BD CSampler Software computer The file is located on the BD CSampler Software CD or flash drive and is on the BD Accuri website www accuricytometers com resources templates NOTE The template is set up for the 24 tube rack only e Open BD CSampler Software e Select File gt Open Workspace or Template e Inthe Open dialog box browse to the location of the bead template file and open the file If adding to an established BD CSampler Software bead file browse to the location of the file i We sah mn h P Au Look In CBD Accuri CSampler ha Ga c fea S SAMPLER ALIGN TEST TUNE 20120402 File Name Sampler 24 tube rack Templete c t Files of Type Workspace and Template c6 c6t cfl cft FEKKIGT 7 Open Cancel 7820099 01 Rev 0 11 BD CSampler Software User Guide 12 Figure 3 1 Open Bead Template Place the following tubes on the 24 tube rack Table 3 1 24 Tube Rack Setup for Validation 2 mL of filtered deionized water according to
58. 5 Be asi C19 c2f c3 f ca c5 coig oa BD gt E1 E2 ha 7 Make a new plot Copy Plots from Collect Aa El ba Plot List suse eal Select a plot and Select a plot and Select a plot and FL1 H sample to analyze sample to analyze sample to analyze Plot 7C FSC H SSC H Plot 8C FL4 H M23 26 0 445 0 M24 1 489 0 4 116 0 3 060 M25 282 548 0 328 24 2355 2 865 12 15 12 15 183 30 47 36 12 97 12 97 2 543 61 16 49 9 98 9 98 306 470 44 1 68 2 2504 350 220 2 oy Figure 6 1 Analyze Tab Workspace The following table describes each of the controls and indicators in the Analyze tab Table 6 1 Analyze Tab Controls Plate Type Disabled in the Analyze tab Plate Name Text box for naming the plate Sample Naming Field Text box for naming the current sample Sample Grid Matrix laid out in the configuration of a multi well plate to organize sample data Each sample has its own well in the Sample Grid The wells are color coded White Does not contain data Blue Contains data Black check mark Currently selected for viewing data Copy Plots from Collect Copies specified plots from the Manual Collect tab See section 6 2 1 for details Plot Controls Set of buttons for creating new plots or overlaying histograms See section 6 2 3 for details All plots created in the Analyze and Manual Collect tabs including o
59. 9 63 522 022 34 1 50 2 173 81 300 479 72 1 72 236 95 158 8 C iv A04 Spk verification 100805 578 898 91 522 310 46 1 53 2 188 34 304 304 68 1 42 237 88 159 2 C i a05 Bok verification 563 712 41 428 335 18 1 82 2 421 27 303 169 39 1 60 177 39 128 8e Zo ks tk 563 661 26 399 766 74 1 83 2 447 65 306 571 81 1 57 178 95 128 46 MM 801 6pk initial 581 321 91 538 205 67 1 48 2 181 10 305 616 95 1 23 233 36 159 82 C Z B02 FL4 Unity 580 580 12 503 150 22 1 47 2 183 73 306 176 29 1 41 238 15 159 92 B 7 503 Spk after unity 2 V BO4 Spk verification c M 805 Figure 3 15 Statistics Tab 8 Peak Bead Data from Successive Days Compare statistics over time for trends or sudden changes in mean values to assess the cytometer s performance The BD Accuri C6 flow cytometer reports arithmetic means Troubleshooting Validation The following conditions may indicate a problem with the flow cytometer or the validation beads e Very broad CV gt 5 0 or multiple populations for FSC H on the 8 or 6 peak beads excluding the doublet population e Fewer than eight peaks for FL1 or FL2 e Fewer than six peaks for FL3 or FL4 To troubleshoot validation e f bead data were not acquired using the slow rate select Slow resuspend the beads and recollect the data e Ifthe beads have been diluted for more than one week kept at room temperature or warmer or ex
60. 96 9 1 Applying MEGA eee 97 92 Viewing VIHTalGanugasasseasrurrraeteseaeee eder 100 9 3 REMOVING VING assein eee eee Aenean Gan 101 APPENDIX A BD CSAMPLER SOFTWARE MENU QUICK REFERENCE 000 2 102 APPENDIX B ADVANGED FLUIDIGS SETTINGS unne 105 APPENDIX C TRACKING USER AC TV ITV ss 107 C 1 Installing the User Tracking Module ccccccccccseceeseeeeeeeeeseeeeesesesseeeeseeeesseeees 107 C 2 Using the Tracking Feature for the First TiIM c ccccccsececseeeeceeeesseeeeseeeeseeeees 108 C 3 Adding Deleting and Modifying User ACCOUNTS rrrrrnrannnnnnnennnnnnnnnnennnnennannennnnen 108 G 3 1 Adding User ACCOUMS ixs2 02 c2s2cc2cieseedenateacndueecassaenteacnducen2sanenteacndeseetencenbtectss 108 C32 Deleting USE ACCOUMS rorarii n jeer estes PP SE 109 Go 35 MANNA PENN NN 109 C 4 Signing In and Signing Out sessioni a a i o 109 C 5 Restoring a Forgotten Administrator Password ccccceeeeeeeeeeeeeeeeeeeeseeeeeseeeess 110 C6 Monitoring USer ACtivity cssccs tencosecosgstcescenccedengsechserwiesstnasserhcdsunessteedierhedemsesadendievee 111 APPENDIX D BD ACCURI G6 ANALYSIS SOFTWARE sunset 112 APPENDIX E SELECTABLE LASER Saa 113 E 1 Installing the Selectable Lasers MOdUIe cccccsecccseeeeceeeeeeeeeceeeeseneeeseeeesaaees 113 E 2 Validating Proper Function After Installation rrrernnrnnnrrnnnrrrnvrrrnrrnnnrennrrnnnrrnnvenn 116 E 3 Annotating Selected Laser Configuration
61. 99 01 Rev 0 BD CSampler Software User Guide Plot 6 404 HPB COS CO x GATE F1in Pi Figure 4 28 Third Plot with Nested Gate Applied R1 in P1 Statistics that reflect the nested gates can be viewed in the Statistics Table Plot 6 A04 HPB CD3 CD4 CD45 CDS Er Volume pL of This Plot of All Mean CD8APC A CV CD3 APC A Median CDS APC A Gated on R1 in F1 SSCS Plot 76 88 00 ooo 333 18528 29 O O S Figure 4 29 Statistics of Plot with Nested Gate e Close the dialog box For instructions on renaming plots and regions and event coloring see APPENDIX F Enhanced Analysis Features 4 8 Moving and Resizing Regions Region Labels can be moved by clicking and dragging the label To move or resize a region e Click on the border of the region Plot 1 401 Unstained GATE No Gating 200 000 400 000 600 000 800 000 ij So0 000 71 000 000 1 600 000 FSC A C62 EEE Figure 4 30 Selected Region in the Batch Analysis Tab e Drag the region to the desired position 7820099 01 Rev 0 45 BD CSampler Software User Guide 4 9 Plot 1 4041 Unstained GATE No Gating 2 i a S oo o o o o F o Ga o Ga 4 500 000 1 000 000 LEDD D D FSC A THE SYKE Figure 4 31 Moved Region in the Batch Analysis Tab Changing the Number of Events in a Plot The number of events displayed in all plots across all samples can be changed in orde
62. AG Aa 81 82 B3 84 B5 m 87 LLI c1 c2 c3 ca cs ce e 500 000 998 644 FSC H oes A E Plot 4 A04 8pk verification run GATE R1 rt A m ne ae Bap A Settings Fluidics Run Unlimited Slow Medium Fast Flow Rate 14 pU Core Size 10 ym Custom Run with Limits Flow Rate 14 ul min 9 Min 0 Sec Set Core Size FR wt Core Size 10 pm Do not collect events outside Threshold Set Threshold 80 000 on FSC H None 100 00 Mi 526 216 0 595 961 0 18 2 42 84 42 Plot 3 A04 8pk ERS ug i of All Set Color Compensation on RI EM SIREN ea ss Plot 18 644 _ Last Run Cumulative Delete Events IV show warning M2 26 0 445 o 2 323 o Events 22 080 M3 1 489 0 4 116 o 2 491 0 00 0 Time 2 00 0 All Bn O Microliters 27 Figure 2 2 BD CSampler Software Manual Collect Tab Workspace Opening a New BD CSampler Software Workspace A new BD CSampler Software workspace Figure 2 2 displays a single FSC A versus SSC A density plot that is pre zoomed to channel values of 1 600 000 and 800 000 respectively Run Settings will be set to Run Unlimited the Threshold will be set to channel 80 000 on the FSC H signal and no color compensation values will be set A new workspace can be used to create an analysis template and to collect a new dataset To open a new workspace e Doone of the following e lf BD CSampler Software is not alre
63. Acquisition settings are saved based on the currently selected sample 4 14 Understanding Fluorescence Spillover Fluorochromes typically emit light over a broad range of wavelengths resulting in the fluorescence signal appearing not only in the expected primary detector for that fluorochrome but in other detectors as well This phenomenon is often called fluorescence spillover and can be a source of confusion when interpreting multi color flow cytometric data 7820099 01 Rev 0 53 BD CSampler Software User Guide 4 14 1 Recognizing Fluorescence Spillover When performing a multi color experiment prepare a set of control samples stained with individual fluorochromes used in the experiment These single stained controls will allow determination of the extent of fluorescence spillover from each fluorochrome The example shown in the figure below shows data collected for a PE Cy7 single stained control Most of the fluorescence signal from PE Cy7 positive cells is detected in the FL3 670 LP as expected However there is also PE Cy7 signal detected in FL1 530 or 533 BP and FL2 585 BP so that plots of data for those detectors appear to have positively fluorescent cells No signal from PE Cy7 appears in detector FL4 ADS HPB CD45 PE Cy A05 HPB CD45 PE Cy AOS HPE CO45 PE Cy Gate PS in alh i Gate PS in all a Gate F3 in alh Ta d E 3 E x 4 T T s Fo e ro 5 a G E 5 E I a m T
64. Colored Events with a Region cccccccccseceeeeeeeceeeeeeeeeeseceseaeeeseeeetaeesseeesaaees 130 Figure F 11 Batch Analysis Tab WOrkSpace cccccccseeeceeeeseeeeaeeeceeeeseeeeaeeseueeseeeseueenseesaes 131 Table F 1 Batch Analysis Tab COntrolS ssicicassnioicedsdindccsnaledseestlackesunlscdevstdeaieoualsddeeshlecsecolacdes 131 Figure F 12 Export Buttons in the Batch Analysis Tab rrrnrrnnrnnnrnnnrnnnrnnnrvnnrnnnrnnnrnnnrnnnnnnnnnnn 132 Panvle G 1s Standardi FCS TAGS sa 133 Table G2 SONS TI NR 135 Table G 3 FCS Tags not in BD CSampler Software Exported Files rrrrnnrenrnrrrrnnrrnnnnrnnnn 135 Table H 1 Example CSV File eee 136 7820099 01 Rev 0 vii BD CSampler Software User Guide 1 INTRODUCTION TO BD CSAMPLER The BD CSampler is an optional accessory for the BD Accuri C6 flow cytometer that allows for the collection of samples prepared in 48 and 96 well plates and standard 12 x 75 mm tubes in a 24 tube rack The BD CSampler is compatible with the following plates 96 well plates standard flat u and v bottom 96 well deep well plates 48 well plates 24 tube rack for standard 12 x 75 mm tubes or microcentrifuge tubes 1 1 NOTE For best results only use the 24 tube rack supplied with the BD CSampler Installing the BD CSampler Install and validate the BD Accuri C6 flow cytometer with 6 and 8 peak beads before installing the BD CSampler Inspect the BD CSampler Accessory Kit content
65. D3 FITC A 61 62 63 64 65 GB 67 Gs 69 610 611612 apap am EC T oH ENE H EE H1 H2 Ha H4 HS HG HF HB HO H10 H11 H12 an e e 2 ked N 3 blue 1 red jo is C6 is connected and ready 2 blue 2 red z C 4blue Select plot type Select plot type Select plot type to make a new plot to make a new plot to make a new plot Ad l i Ad el l Ad l i Threshold Set Threshold All 231 332 0 100 00 100 00 413 478 48 104 468 97 65 92 124 17 292 769 5 i 80 000 on FSC H one Run Settings Fluidics Run Unlimited Slow Medium Fast GR e R Flow Rate 66 pL min Taj Core Size 22 pm Iv 200000 events Custom PP minfo Sec Do not collect events outside Flow Rate 11 ul min ia Plot 2 A03 HPB CD3 CD 45 Count Volume pl ofThisPlot of All Mean CD3FITC A CV CD3FITC A Median CD3 FITC A All 231 332 0 100 00 100 00 11 065 74 135 24 1 610 0 I Plot 3 A03 HPB CD3 CD45 Count Volume pL of This Plot of All MeanFSC A CVFSC A Median FSC A Set Color Compensation All 231 332 0 100 00 100 00 413 478 48 65 92 292 769 5 Last Run Cumulative Delete Events V show warning a Events 231 332 0 00 0 Time 000 0 All OD Microliters o Outside v 0 Events Sec 1 Bevarte ku ay Data Capacity Used 1 of 96 000 000 Events m Figure 4 1 BD CSampler Software Manual Collect Tab The following table describes each of the controls and indicators in the Man
66. Fax 64 9 571 2469 bd anz bd com Japan Nippon Becton Dickinson Toll Free 0120 8555 90 Tel 81 24 593 5405 Fax 81 24 593 5761 Europe BD Biosciences Tel 32 2 400 98 95 Fax 32 2 401 70 94 bdbiosciences com eu help biosciences europe bd com United States BD Biosciences Toll Free 877 232 8995 Fax 800 325 9637 bdbiosciences com answers bd com For Research Use Only Not for use in diagnostic or therapeutic procedures Class 1 Laser Product BD Accuri BD CSampler Software BD CSampler VirtualGain BD Accuri C6 Flow Cytometer and Science is hard Flow cytometry should be easy are trademarks of Becton Dickinson and Company 2012 7820099 01 Rev 0 137
67. First Gate for Creating Nested Gates Drawn in Plot 4 42 7820099 01 Rev 0 BD CSampler Software User Guide e Create a new plot and click on the GATE button to apply the P1 parent gate Change Gating for Plot 5 1 FSC AISSC A 1 on all events FSC 4 55C 4 Clear All Figure 4 24 Applying the Parent Gate to Plot 5 e Close the dialog box The plot displays only the populations within the parent gate Plot 5 404 HPB CD3 CD GATE P1 4 CD3 FITC A got A Figure 4 25 Parent Gate Applied to Dot Plot 7820099 01 Rev 0 43 BD CSampler Software User Guide 44 In the plot that is gated on the parent gate plot 5 in this example draw a second region or marker around a subset of the population displayed in the plot R1 Plot 404 HPB COS CO GATE Fi ai ur CDS FITC A ID s2 amp a Figure 4 26 Second Gate for Creating Nested Gates Create a third plot and click on the GATE button In the Change Gating dialog box select the option in which the second gate is in the parent gate e g R1 in P1 see Figure 4 27 Alternatively selecting the on all events option e g R1 on all events un nests the gates Change Gating for Plot 6 Gating choices Include E PLIFSC AJISSC A E Fi on all events FSC ANSSC A W Ri inPl FLL AJSSC A R1 on all events FL1 4 55C 44 Clear All Figure 4 27 Applying the Child Gate Apply the gate This is the child gate 78200
68. Fluidics section of the Auto Collect tab 5 6 2 Setting the Threshold Use thresholds to gate out light scatter and or fluorescence signals caused by debris in cell samples and electronic noise inherent in the system When set correctly for any given sample set resolution of particle or cell light scatter and fluorescence signals is greatly improved and data set size often can be reduced By default BD CSampler Software is set to a primary threshold of channel 80 000 on FSC H For more information on setting thresholds see section 4 2 2 5 6 3 Setting a Run Limit The run limit defines when data acquisition will stop The following parameters can be used individually or in combination to set a run limit e Time e Volume e Number of events in a specified gate If multiple run limits are set data collection stops on whichever limit is reached first NOTE If run limit settings were modified in the Manual Collect tab they do not carry over in the Auto Collect tab For more information on setting run limits see section 4 2 6 5 6 4 Agitating Samples The agitate feature is designed to keep samples in uniform Suspension during data collection Agitate gently shakes the sample plate tube rack in 15 second cycles BD CSampler Software can automatically perform one two or three agitation cycles from the Auto Collect tab during the plate run The user can specify whether to agitate after a given number of samples or in one minute i
69. HRESHOLD Permanently eliminate events on FSC H less than S0000 Range Minimum 10 SECONDARY THRESHOLD Ely eliminate additional events on less than Range Minimum 10 f Only this sample Apply Close f All samples Figure 4 4 Primary Threshold Drop Down List e Type 80000 in the less than edit box to set the threshold minimum to channel 80 000 NOTE A lower or higher FSC H threshold may be required when working with small cells such as platelets or bacteria or large cells Such as cell lines respectively Refer to Technical Note Threshold and Analysis of Small Particles on the BD Accuri C6 Flow Cytometer available at http www accuricytometers com resources application notes or Table 4 2 To apply a secondary threshold select the threshold parameter from the Secondary Threshold drop down list Type a value in the ess than edit box to set the threshold minimum e Doone of the following o Select the Apply to All samples radio button to apply settings to all samples including all previously collected data in other data wells o Select the Apply to Only this sample radio button to apply settings to the current sample only Threshold Settings for Unstained PRIMARY THRESHOLD Permanently eliminate events on FSC H less than S0000 Range hinimum 107 OPTIONAL SECONDARY THRESHOLD Permanently eliminate additional events on less than Range k nimum 10 Apply to Only this sam
70. Table 5 2 Plate Setup for Ending a Data Collection SESSION cccccceeccseeeeeeeeeeeeeseeesaeeeneees 78 Figure 5 15 Export Sample SeninGS sarissa a ae aa E a N N a 79 Figure 6 1 Analyze Tab Workspace ccccccccseeceececsececeecceucecaueeceeeceuceseueeseessueesseessaeessusenanss 81 Table 6 LANE ETA COMMONS SS 81 Figure 6 2 Selecting Plots to Copy from the Collect Tab rrrrrnnnnrnnnnnnnnnrvnnnrrnrnnrennnnrnnnnrrnnnsen 82 7820099 01 Rev 0 V BD CSampler Software User Guide BD CSampler Software User Guide Figure 6 3 Plot List Containing Copied Plots rrrrrrnnrrnnnnrnnnnnrnnnnennnnrnrnnnnnnnnrnrnnnnnnnnennnnsennnnen 83 Figure 6 4 Blank Overlay Histogram PlOt cccccccsececeeeeeeeeseeeeseeseeeeseueeseeseueeseeeeseeesaneeneees 84 Figure 6 5 Overlay Histogram Plot with Data rranrrnnnrnnnnonnnnnnnnennnnennnnnnnnennnnennrnnnnennnnennnennnee 84 Figure 6 6 Overlay Histogram LeEQend cccccccsecccseeeceeeceececeueeceeeceuceseueeseeessusesseesseeessueenaees 85 Figure 6 7 Overlay Histogram Legend with Color Palette ccccccceeeeseeeseeeeseeeeseeeseeeeseees 85 Figure 6 8 Analyze Tab Plots with Gating Applied rrrrerrnrrrrnnrerrarerrnnrernnnerrnnnennanenvnnnennnnene 86 Figure 6 9 Analyze Tab Two Samples with the Same PIlots ranrrennnrnvnrnnrrennnvrvnnnnvrenrnnrrennn 86 Figure 7 1 Statistics Tab WorkSpace ccccceecceeeeeeeseeeeeeeeceueeseeeeeeeseueeseeseu
71. ady open double click on the BD CSampler Software icon on the computer desktop e If BD CSampler Software is already open select File gt New Workspace File If desired save any unsaved changes to the previous workspace when prompted Loading and Ejecting a Plate BD CSampler controls movement of the plate platform into position for efficient loading or ejecting racks and plates To move the BD CSampler arm to the load or eject position click on the Load Plate Eject Plate toggle button 7820099 01 Rev 0 BD CSampler Software User Guide 2 9 2 6 7820099 01 Rev 0 NOTE After 15 minutes in the eject position the arm and plate platform automatically return to the home position with the SIP in the wash station the white receptacle on the plate platform Aligning the BD CSampler after a Collision The BD CSampler performs an alignment to verify that the plate is aligned to the SIP every time the flow cytometer is powered up or if the BD CSampler arm collides into an object A manual alignment can be performed in BD CSampler Software at any time If there is an obstruction in the path of the BD CSampler arm BD CSampler Software displays a red Traffic Light and opens a message box indicating that a collision has occurred To perform an alignment e Remove any objects from the BD CSampler mat Do one of the following e Ifa collision has occurred click on the Align button in the Collision Detected dialog box e Ifa
72. al Collect and Analyze tabs that are available to preview Table See section 7 2 for details See section 7 2 for details Creating the Master Statistics Table The Master Statistics Table enables creation of a customized data table for multiple samples within a given BD CSampler Software file Statistics Column Selector Selects the data to view in the Master Statistics Table for each sample See section 7 2 for details To create the Master Statistics Table In the Statistics Column Selector enable the check boxes under the data items to view per plot BD CSampler Software automatically adds columns to the Master Statistics Table Display Statistics Column Selector Plot Preview Add columns to your master statistics table by selecting a cell Plot 1 FSC A SSC A Plot 1 FSC A SSC A Volume ul Events uL of This Plot of All Mean FSC A Mean S5C A CY FSC A CV SSC A Plot 2 FL1 A v DD DD END ee Na DN EN GA GE o Plot 3 FSC A Plot 2 FL1 A Volume ul Events uL of This Plot of All Mean FL1 4 Soe i v EE ee os VEN GE ee ee ee Plot 3 FSC A Volume uL Events uL of This Plot of All Mean FSC A CVWFS5CA f Poa dM O d M Vo on On Master Statistics Table Select cells from the Sample Selector and Statistics Column Selector to build your table Plot 2 FL1 A Plot 3 FSC A Events ful CWFSC A CWSSC A Events ul CVFLI A Events ul Mean FSC A Plot 1 FSC AJ SSC A Figure 7 2 Crea
73. alog box BD CSampler Software automatically displays the percentage of events within the region Flot 1 201 Unstained pl GATE No Gating E 200 000 434 715 FSC A aar SEK Figure 4 18 Using Polygonal Gating Tool To create a vertical marker in a histogram plot Click on the Vertical Marker Tool 7820099 01 Rev 0 39 BD CSampler Software User Guide e Click the cursor at the point along the x axis on which to place the marker BD CSampler Software automatically displays the percentage of events to the left V7 L and right V7 R of the marker Plot 2 401 Unstained GATE No Gating od o o Figure 4 19 Using the Vertical Marker NOTE Only a single vertical marker can be set per histogram To create a horizontal marker in a histogram plot e Click on the Horizontal Marker Tool e Click and drag the cursor horizontally across the area to apply the gate BD CSampler Software automatically displays the percentage of events within the margins of the marker labeled M7 Flot 2 404 Unstained x GATE No Gating 10 000 12 800 Figure 4 20 Using the Horizontal Marker 40 7820099 01 Rev 0 BD CSampler Software User Guide 4 7 2 Applying a Gate to a Plot e Click on the GATE button at the top of the plot Only polygon P rectilinear R and marker M gating regions automatically appear in the Gating dialog box list of options e To include vertical markers or quadra
74. always 32 since integers are stored in 32 bits in Java range for all parameters is always 16777216 PnN The name of parameter N Unchangeable Parameters are the default values from BD CSampler Software PnE For parameter N this denotes if Unchangeable linear or logarithmic amplifiers are used It is always 0 0 because linear values for data are always saved This is an optional tag 7820099 01 Rev 0 133 BD CSampler Software User Guide The name of the fluorescence Rename parameter dialog stain or probe used for parameter N This tag is used for the custom parameter name TOT Number of objects stored in the Determined by the amount of data list The cumulative event events acquired by user total for the sample DATE The date the represented Defined by the beginning time sample was last acquired into of the most recent acquisition DD MMM YYYY Set by the computer s clock SCYT The name of the cytometer used Unchangeable for the measurement It is always BD Accuri C6 SPILLOVER The standard tag for color Determined by values entered compensation into Color Compensation dialog STIMESTEP Hard coded value of 0 1 which Unchangeable is in Seconds Represents the name of the Saving as a different file workspace which is also the importing an FCS file or name of the c6 file before the exporting an FCS file would c6 extension If exporting an change this value FCS file this value is the name of the
75. ard Install the MOUIE cccccccceeeeceeeeeeeeceeeeneeesaueeseueeseeseeeesaeeeaes 115 Figure E 4 Install Wizard Setup Completed Successfully rrrarrrrnnrrrrnrrvrnnrrrrnnrennnrennnnrennnr 115 Figure E 5 Selectable Laser Controls in the BD CSampler Workspace rrrrnnnnnnnnnnnrrnvnnnnen 116 Figure E 6 Selectable Laser Options 3 Blue 1 Red rrrnnnernnnrnrnnnennnnrnrnnrennnnrnrnnrennnnennnnennnnne 116 Figure E 7 Selectable Laser Options 2 Blue 2 Red rrrnnnennnnenrnnnennnnrnrnnrernnnenrnnrennnnennnnennnnne 117 Figure E 8 Selectable Laser Options 4 BIue r rrrrrrrarrnrnnnrvrnnrnvannrrnnnrnranrennnnenranrnnnnnennanennnnne 117 Figure E 9 Evaluation of 6 Peak Validation Beads for the 2 Blue 2 Red Configuration 117 Figure E 10 Evaluation of 8 Peak Validation Beads for the 4 Blue Configuration 118 Figure E 11 Renamed Data Well to Indicate 4 Blue Configuration ccccccesseeeeeeeeeeeeees 119 Table E 1 3 Blue 1 Red Configuration 1 Standard Filters rrnrrnnnnnrrnnnrnnnnnennnnrnnnnrennnnrnnnn 119 Table E 2 3 Blue 1 Red Configuration 2 rrrnnnrnrnnnrrnnnrrrannrnnnnenrnnrnnnnnennnnrnnnnnennarnnansennansnnnn 120 Table E3 2 BIE 2 RAET CONIG UA ON ae 121 Table E 4 4 Bide CGoniguralion Lauvussvss5r ves svarene 121 vi 7820099 01 Rev 0 BD CSampler Software User Guide Table E 5 4 BUE COnliGuraniOn 2 Lavere 121 Table E 6 4 Blue GONTIGULATON B sesaiesc
76. ase of the SIP To perform a backflush e Doone of the following e Click on the Backflush button in the Manual Collect tab e Select Instrument gt Run Backflush Cycle e Wait for the BD CSampler to move to the Eject position and place a container under the SIP e Click on the Backflush button in the Run Backflush Cycle dialog box Run Backflush Cycle Place appropriate container under SIP to catch Fluid Cancel Figure 8 1 The Run Backflush Cycle Window Cleaning the Flow Cell Clean the flow cell as a part of regular maintenance or to correct performance issues of the cytometer 8 2 1 Running the Unclog Cycle The Unclog Cycle purges the flow cell of debris To purge the flow cell e Doone of the following e Click on the Unclog button in the Manual Collect tab e Select Instrument gt Run Unclog Cycle e Wait for the BD CSampler to move to the Eject position and place a container under the SIP e Click on the Unclog button in the Run Unclog Cycle dialog box 7820099 01 Rev 0 91 BD CSampler Software User Guide 8 3 92 Run Unc log Cycle Place appropriate container under SIP to catch Fluid Cancel Figure 8 2 The Run Unclog Cycle Window 8 2 2 Running an Extended Clean of the Flow Cell During the Extended Flow Cell Cleaning Cycle the flow cell fills completely with the extended flow cell cleaning solution from a sample tube on the SIP This cycle automatically shuts down the flow cyt
77. ast action that was performed in n BD CSampler Software Not all actions are undoable Reverses an Undo action Copy Copies a selected marker or region from a plot or selected statistics from the tables in BD CSampler Software Pastes copied markers and regions into new plots Allows individual parameters in either the current sample or all samples to be renamed Display Opens a dialog to change the number of events displayed in all plots Auto Select next well Opens a dialog to configure whether BD CSampler Software automatically selects wells vertically horizontally or not at all at the completion of each sample collection Removes all VirtualGain settings from the entire BD CSampler workspace Hides shows the median statistics in the Statistics Table Export Sample Settings Instrument Set threshold Opens the Threshold dialog box for setting the ms primary threshold value and setting an optional secondary threshold Opens the Compensation Settings dialog box for correcting fluorescence spillover Runs a cleaning fluid cycle Run Decontamination Fluid Runs decontamination fluid cycle Cycle Runs the unclog cycle to clean the flow cell Runs the backflush cycle to clean the SIP and remove clogs at the base of the SIP Cleans the flow cell for an extended time Aligns the BD CSampler arm to the SIP Calibrate Fluidics Initiates fluidics calibration to ensure that the BD Accuri C6 provides accurate volume
78. at displays the Height signal for the threshold channel and observe the effect on data as the threshold is raised or lowered Table 4 2 Suggested Starting FSC H Threshold Settings for Various Cell Types Log scale is helpful Log scale is helpful Unfixed freshly isolated 200 000 to 500 000 Linear or log scale display of FSC cells white blood cells SSC spleen thymus Fixed cell suspensions 200 000 to 500 000 Linear or log scale display of FSC white blood cells spleen SSC thymus SSC Bacteria microparticles Dual thresholds See document titled Threshold and suggested Analysis of Small Particles on the BD Accuri website CAUTION Take care when setting the thresholds before or during data collection Any event not meeting the threshold criteria will be not be acquired or saved When changes are made to the threshold values after data collection BD CSampler Software displays a warning message if the new threshold value will result in permanent data loss Warning This will PERMANENTLY DELETE all events and data from this sample Figure 4 3 Threshold Settings Warning Message To set the threshold e Doone of the following Select Instrument gt Set threshold Click on the Set Threshold button in the Instrument Control Panel e Select the primary threshold parameter from the Primary Threshold drop down list in the Threshold Settings dialog box 7820099 01 Rev 0 27 BD CSampler Software User Guide PRIMARY T
79. ata in real time during the collection The flow cytometer stops sampling from the tube or well when the run limit is reached 7820099 01 Rev 0 33 BD CSampler Software User Guide 4 3 34 CAUTION If the ADD TO button is selected BD CSampler Software will collect data into a well that already contains data 4 2 10 Pausing Data Collection Sample acquisition can be interrupted any time during a run To stop a run e Click on the PAUSE button To restart the run e Click on the ADD TO button BD CSampler Software resumes data collection in the current well Ending a Data Collection Session When sample collection is completed clean the SIP and fluidics lines by following the procedure below e Load a 24 tube rack containing the following tubes Table 4 3 Plate Setup for Ending a Data Collection Session 2 mL of filtered deionized water Empty data well for example D4 2 mL of decontamination solution PN Empty data well for example D5 653154 2 mL of filtered deionized water Empty data well for example D6 e In BD CSampler Software select the well corresponding to the first tube of water Set the time limit for two minutes and the fluidics speed to fast e Click on the RUN button e In BD CSampler Software select the well corresponding to the decontamination solution e Set the time limit for two minutes and the fluidics speed to fast e Click on the RUN button e In BD CSampler Software select the
80. ble on the BD CSampler Software CD or flash drive e Number of peaks e Mean channel numbers for the top peaks and forward scatter e CVs for the top peaks and forward scatter e If this is the first validation run send a copy of the file to Accuri Technical Support for analysis See Section 8 6 for email address 3 4 Monitoring Validation Bead Data If a single BD CSampler Software file is used to collect validation bead data it is easy to monitor flow cytometer performance over time using the Statistics tab To monitor the bead data e Save the 8 and 6 peak validation bead data from each day in separate wells Grouping the 8 peak data runs separately from the 6 peak runs in the 24 well grid simplifies use of the Statistics tab 7820099 01 Rev 0 19 BD CSampler Software User Guide 3 9 20 e Create a table in the Statistics tab that contains the mean channel numbers and CVs for the top peaks on each detector and the forward scatter see chapter 7 for details on creating tables in the Statistics tab BD CSampler BD Statistics Statistics Column Selector Add columns to your master statistics table by selecting a cell Plot 1 FSC HISSC IC Volume Events of This Mean F Mean 5 CV FSC H CV 5 Median F Median 5 E VE es se ee a a a a a a a e e a a Plot 1 No Sample Gate No Gating Display Plot Preview Plot 1 FSC H SSC H
81. blue configuration with a 630 30 in FL4 to detect PE TR 7820099 01 Rev 0 BD CSampler Software User Guide Table E 3 2 Blue 2 Red Configuration FL1 530 30 FITC GFP CFSE or 533 30 FL2 585 40 PE PI PE Texas Red FL3 780 60 APC Cy7 and equivalents FL4 675 25 APC and equivalents Table E 4 4 Blue Configuration 1 FL1 530 30 FITC GFP CFSE or 533 30 FL2 585 40 PE PI PE Texas Red FL3 780 60 PE Cy7 and equivalents FL4 675 25 PerCP Cy5 5 PE Cy5 Table E 5 4 Blue Configuration 2 FL1 530 30 FITC GFP CFSE or 533 30 FL2 585 40 P FL3 780 60 PE Cy7 and equivalents 630 30 PE Texas Red 7820099 01 Rev 0 121 B D CSampler Software User Guide Table E 6 4 Blue Configuration 530 30 FITC GFP CFSE or 533 30 675 25 PerCP Cy5 5 PE Cy5 630 30 PE Texas Red E 5 Selectable Laser Application Examples E 5 1 2 Blue 2 Red Configuration Examples Table E 7 Detector and Filter Configuration for 2 Blue 2 Red Examples 1 and 2 530 30 FITC or 533 30 780 60 APC Cy7 122 7820099 01 Rev 0 BD CSampler Software User Guide Example 1 2 Blue 2 Red for Simultaneous FITC PE APC and APC Cy7 The following images illustrate a 4 color analysis of CD3 CD4 cells Human peripheral blood HPB was stained with CD3 APC Cy7 CD4 APC CD45RA FITC and CD45RO PE 3 Light Scatter Gate F1 Gate P1 3 8 7 3 or d q 33 Se ef 2 a me 3 r r 0 1 0
82. cles if applied before stopping the plate run The user can eject the plate when the run is stopped The settings in Auto Collect cannot be changed e Abort WelK Immediately stops data acquisition stops the fluidics and ejects the plate Settings cannot be changed from the Auto Collect tab in the aborted state To stop acquiring data after it has started e Click on one of the following buttons e INTERRUPT PLATE e ABORT WELL To restart a run during an Interrupt state e Click on the AUTORUN button BD CSampler Software continues acquiring data where it left off To restart a run during an Abort state e Doone of the following e To begin collecting samples at the next well click on the Autorun button e To collect from the same well close the run display on the Auto Collect tab click on the Manual Collect tab and run the sample manually 5 8 3 Viewing Sample Plots All plots created in the Manual Collect tab are listed in the Select Plot drop down list beneath the plot window They can be selected at any time during data collection Two plots can be viewed at a time NOTE Plots can only be added modified or deleted from the Manual Collect tab A03 Events are being recorded T das ie daryi i Figure 5 14 Run Display Viewing Sample Plots To view plots e Select plots to view from the Select Plot drop down list under each plot corral 7820099 01 Rev 0 BD CSampler Software User Guide 5 9 En
83. collision did not occur select Instrument gt Align BD CSampler x CSampler has detected a collision You must align the CSampler to continue collecting This may take a few minutes Align Figure 2 2 Collision Detected Window If a second collision occurs BD CSampler Software automatically performs a second alignment If the second alignment fails contact Technical Support Samples left on the sample stage can be recovered by gently pushing down on the white cylindrical motor housing Exiting BD CSampler Software e Select File gt Quit e f prompted to save changes to the BD CSampler Software workspace do one of the following a Click onthe Yes button to save changes Click on the No button to close BD CSampler Software without saving changes c Click on the Cancel button to cancel the exit and keep BD CSampler Software open BD CSampler Software User Guide 2 7 10 Using the Example BD CSampler Software File An example BD CSampler Software file of a four color analysis of human peripheral blood HPB 4 Color Example c6 can be downloaded from the BD Accuri website www accuricytometers com resources tutorials This data file can be used to explore various tools in BD CSampler Software without the worry of corruption or loss of experimental data Figures throughout this user guide show data from the example file To create the example file four sample tubes were used to assess the CD3 CD4 and CD3
84. d Sample from the associated drop down list Run Settings C Run Unlimited Run with Limits M 50000 events in Ungated Sample lo Min 0 See lo uL Figure 3 4 Run Limits 50000 Events If desired click on the Agitate button to resuspend the beads in the 24 tube rack Select the Slow radio button in the Fluidics section of the Control Panel Click on the RUN button to start acquisition Acquisition automatically stops after 50 000 total events are acquired CAUTION Make sure the well in BD CSampler Software is empty before starting the run If the button displays ADD TO the well already contains data NOTE The R1 region may not encompass the main population of bead events on the FSC H vs SSC H plot This is common and acceptable at this stage Name the sample by typing a name in the text box just above the Sample Grid Include the date in the sample name to differentiate it from samples collected on other dates NOTE Samples can be named before during or after collection A01 8 Peak Beads 20090218 Figure 3 5 Sample Name 8 Peak Beads When the collection is finished click on the Wash button to minimize sample carryover Run 6 Peak Validation Beads If desired click on the Agitate button to resuspend the beads in the 24 tube rack Click on the well corresponding to the tube containing 6 peak beads 7820099 01 Rev 0 13 BD CSampler Software User Guide 10 11 12 AID Add AZ B10 B11 Bi C10 C1
85. d on a BD Accuri C6 flow cytometer can be imported into BD CSampler Software Export FCS File Exports and saves the currently selected data well as an FCS 3 0 file to a specified folder Exported files are compatible with off line analysis programs such as FCS Express FlowJo and WinList Export ALL Samples as FCS Saves all of the data wells as individual FCS 3 0 files in the folder FCS Exports onthe computer desktop Exported files are compatible with off line analysis programs such as FCS Express FlowJo and WinList Export ALL Samples to Third Exports and saves all data wells as individual Party FCS 3 0 files that enable autoscaling in third party applications such as FlowJo Export Plot Data as CSV Saves an individual file in csv format for further analysis in spreadsheet programs All data for every event in the selected plot is exported See APPENDIX H for an example csv file 7820099 01 Rev 0 BD CSampler Software User Guide Exports sample settings as a csv file for viewing data in a spreadsheet Sample settings include acquisition criteria sample names parameter names and compensation values File cont Set Plot Drag and Drop Available with Enhanced Analysis Features Format Allows plot format to be toggled between png low resolution or eps high resolution Prints the selected plots and associated Statistics Quits BD CSampler Software and closes the application Edit Undo Undoes the l
86. d take this into account and the average volume in the sample tube during the acquisition should be used For example if 100 uL are to be acquired from a 1000 uL sample the calibration volume would be 950 uL The average volume Starting Volume Ending Volume 2 during the acquisition is 1000 900 2 The cytometer will perform a calibration cycle taking approximately 13 minutes Once completed the traffic light will revert to green with the C6 is connected and ready status message Repeat fluidics validation using a reference count bead as described above If calibration fails BD CSampler Software will display a message that indicates that instrument calibration has failed Troubleshooting Instrument Calibration e Make sure that the calibration tube did not run dry during calibration e Repeat calibration preparation routine including running the decontamination cycle 70 ethanol and water e Repeat the calibration with a new calibration sample e If calibration fails a second time replace the peristaltic pump tubing in the cytometer and repeat the preparation calibration routine e If calibration fails a third time contact Accuri Technical Support The cytometer will still operate normally after a failed calibration However the volume measurements for the desired samples may be incorrect because the cytometer will revert to the factory set default fluidics calibration settings All other aspects of the data will be nor
87. ding a Data Collection Session Upon completion of each run it is recommended to clean the SIP and fluidics lines using the following protocol e Load the tube holder with the following tubes 2 mL of filtered deionized water Empty data well for example H10 2 mL of decontamination solution PN Empty data well for example H11 653154 2 mL of filtered deionized water Empty data well for example H12 e In BD CSampler Software select the well corresponding to the first tube of water Make sure the well contains no data e Set the time limit for two minutes and the fluidics speed to fast e Run the sample e In BD CSampler Software select the well corresponding to the decontamination solution Make sure the well contains no data e Set the time limit for two minutes and the fluidics speed to fast e Runthe sample e In BD CSampler Software select the well corresponding to the second tube of water Make sure the well contains no data e Set the time limit for two minutes and the fluidics speed to fast e Runthe sample e When the run is finished eject the plate tube rack 5 10 Saving a BD CSampler Software File Always save BD Accuri C6 data as a BD CSampler Software file c6 A BD CSampler Software file is a comprehensive and often large data file that contains instrument settings FCS files and plot layouts For details on saving a BD CSampler software file see section 4 12 5 11 Creating a BD CSampler Sof
88. e BD CSampler Software File e If necessary navigate to the location to save the file e Inthe Save dialog box enter the file name and click on the Save button The file is saved with the extension c6 Creating a BD CSampler Template A BD CSampler template contains a predefined workspace for quick and easy setup and analysis All markers regions gates parameter names and sample names are saved without any data points BD Accuri provides several templates see the BD CSampler Software CD flash drive or the BD Accuri website at www accuricytometers com resources templates or create custom templates To create a template e Define plot gating and acquisition settings in a blank workspace or use the current c6 file e Select File gt Save template as 7820099 01 Rev 0 BD CSampler Software User Guide gt o Save Save In C CFlow Sampler v 3 ActivationKeys CJ icons CJjre1 6 0 05 J libraries Accuri_CFlow_Sampler_Bead_Template_24 Tube_Rack New CFlow File Template File Name Te st Samples te mplate Files of Type CFlow Template c6t v Cancel Figure 4 43 Save BD CSampler Software Template e If necessary navigate to the location to save the template file e Inthe Save dialog box enter the file name and click on the Save button BD CSampler Software saves the file with the extension c6t NOTE
89. eate the histogram of interest See section 6 2 for details e Copy plots from the Manual Collect tab see section 6 2 for details Analyze ained A B C D E F G H Make a ne o a Copy Plots from Collect Ad JE Figure 9 2 Setting Up Histograms for VirtualGain Apply the appropriate gating to the plots in the Analyze tab Do one of the following e Select a histogram plot from the sample to which the other samples will be aligned This sample is the standard sample e Select an empty well to align data to a specific channel instead of a collected sample Click on the x axis label on the standard sample and select Virtua Gain from the pop up Parameter List In the VirtualGain dialog box do one of the following e Move the peak definition marker vertical line in the Standard Sample plot to the center of the peak that will be the reference point Other samples will be aligned to this position e lf an empty well was selected in step 3 move the peak definition marker vertical line to the channel to assign as the reference point 7820099 01 Rev 0 97 BD CSampler Software User Guide Standard Sample Peak definition marker Figure 9 3 Aligning Plots e If needed use the Zoom Tools in the Analyze tab to change the zoom level in the VirtualGain dialog box e Click on the small sample grid icon in the center of the Sample to Align plot e Open the sample to be
90. eecptatastsklendctnbeadeosnlecgsenteutceicidensedentcesniewicarhiuciecontande 122 Table E 7 Detector and Filter Configuration for 2 Blue 2 Red Examples 1 and 2 122 Figure E 12 Gating Example Using the 2 Blue 2 Red Configuration rarnrrnnrnnnnrnnvrnennnnnnn 123 Figure E 13 BD Cytometric CBA 30 Plex Bead Mixture collected on the BD Accuri C6 run in 2 Blue 2 Red mode using the Selectable Lasers Module PN 653126 cccseceseeeseeeneeenes 124 Table E 8 Detector and Filter Configuration for Selectable Lasers 4 Blue Example 1 124 Figure E 14 4 Blue Configuration and the Optional 780 60 Optical Filter at Detector FL3 125 Table E 9 Detector and Filter Configuration for Selectable Lasers 4 Blue Example 2 125 Figure E 15 HPB Stained with CD45 FITC CD4 PE CD8 PE Texas Red and CD3 PE Cy5126 Figure F 4 Creating a Live Gate rrrrrannrnnnnrnrannrnnnnenrnnnnnnnnennanennannennnnennnnsnnnnnennansnnnnnennnnsnnnnne 127 Figure F 5 Enable Renaming of the PIlot rrnnnrnnnrrnnnevnnrnnnnennnnennnnnnnnennnnennrnnanennnsnnnsennnsennn 128 Figure F 6 Type a New Plot Name rrannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnsnnnnsnne 128 Figure F 7 New Plot NaMe sicsiuisciossncncatacien che annsecns aie pchaanne aa che anna ect hia a aE ndi 128 Figure F 8 Select a Region to Color scannen A aE 129 Figure F9 Selecta GOlOn Or REGION sa 129 Figure F 10
91. eeeseeeetaeeeeseeeeeaaees 101 Figure 9 9 Toggle between VirtualGain Applied Left and Not Applied Right 06 101 Figure B 1 Custom Radio Button rarrnnnrrnnnernnnrvnnrnrnnennnrennnennnnennnnnnanennnnennnennanennnsennsennnsennn 105 Fiqur B2 Sel COE SZE uavne Re GAN AN RS 105 Table B 1 Core Size and Flow Rate Guide rrrrrranrrnnnnrnrannrnnnnrnranrnnnnnennnnrnnnnnennanrnnnnsennnnsnnnn 106 Figure C 1 User Tracking Installer ICON rrrannrrnnnrrrnnrrrnnnerrnnrnranrernnnrnrnnrennnnennnnsennnnennnssnnnnne 107 Figure C22 Installation Dialog BOX ass 107 Figure C 3 Username and Password Dialog BOX cccccsscccceeecceeeeeeeeeesaeeeeseesesaeesaeeeesaaees 108 Figure C 4 The Users Information BOX cccceccccseeeeceeeeseeeeececeseeeeeseusessueeeseeeesseeesseeesaaees 108 Figure C 5 Adding a NOW WSe lsc scccicin cecicksxtecdcan PA ea eden redde 109 FOUE C 6 DENN APS 109 Figure C 7 Username and Password Dialog BOX rrrarrrrnnnrnnnnrnnnnnennnnrnrnnrnnnnnennnnrnnnnnennnnnnnnne 110 Figure C 8 userUsage Log are 111 Figure C 9 CytometerSupportFiles Folder rrrrnrrrnnnrrnrnnnevnnnenrannrnnnnrnrnnnnnnnnennansennnnennansennnne 111 Figure E 1 Selectable Lasers Installer rrrarrrnnnnrnranrnrnnnrrnnnrnrannrnnnnrnrannnnnnnennansnnnnnennanennnnee 113 Figure E 2 Install Wizard Choose Activation Keys Directory ccccsecccseeeceeeeeeeeeeeeeneeeeaes 114 Figure E 3 Install Wiz
92. er cccccccccsececssececeeeeeseeeeseeeesseeeeseueetseeesees 53 4 14 1 Recognizing Fluorescence SpilloVer cccccscscecseeeeseeeeceeeesaeeeseneesaeeesees 54 4 14 2 Correcting Fluorescence SpillOVer cccccccccceececsececeeeeceesesaeeeeseeeesseeesees 55 4 14 3 Troubleshooting Color Compensation arrrnnnnrnnnnnnnnnnrnnnnrnnnnrrnnnnrnnnnrennnernnn 58 AAS GHANGIAG PaAValnCle lS messi snscsbsccas dese vensece sean sicat a 60 216 AAN STE NN 60 Ale Copying ana Pasing PIOUS Haave 60 ANS Printing Daldinia eee 61 219 EXDORINO and IMPOring FES sr 62 AUTOMATIC DATA ACQUISITION sesscucsoeshianessaccadhecn ease 64 5 1 Viewing the Auto Collect Tab rrarrrrnnnrnnanrvrnnnrnrannnnnnnennanennnnnrnnnnennnnsrnnanennansennnsennnn 64 5 2 Running a Sample Plate in Auto Collect rrrnrnrnnnnrrvnnnvnnnnrnvnnnvnnnnrnvnnnvnnnnrnvnnnnnnnnennnn 66 53 ASSAM a Plite T PE Lose anden 66 54 NEMNE P ua esdee 67 55 USING Sampe SEIS irana A ONA 68 5 5 1 Creating Sample Sets arrien A E E 68 5 5 2 Viewing Sample SettinGS cccccccsscccsseeeceeceeeeeeeceeeesseeeeseeeesseeeeseueesseeeesees 70 5 5 3 Modifying Sample SettingS sarera aaa a a 70 5 54 SAN a SaMPE S PSR 71 555 ROSE HR 71 5 6 Defining Data Acquisition Settings rrrrarrrnnnnrrrnnrnrnnnrnrnnrnnnnnrnnnnrnnnnnennanennnnnennnsennnn 71 5 6 1 Setting the Fluidics Rate cccccccecesseccceeeccececseeccecesseecceeecceeeseeesceeesa
93. er Open the userUsage log file 7820099 01 Rev 0 Text Document 2012 04 03 File 2012 04 04 File File Text Document File Text Document Microsoft Excel C 111 BD CSampler Software User Guide APPENDIX D BD ACCURI C6 ANALYSIS SOFTWARE The optional BD Accuri C6 Analysis Software allows analysis of BD Accuri C6 Software files on a computer that does not have a connection to the BD Accuri C6 flow cytometer BD Accuri C6 Analysis Software is available in two forms e PN 653122 BD Accuri C6 Analysis Software for PC or Mac e PN 653123 BD CSampler Analysis Software for PC or Mac BD CSampler Analysis Software has all the functionality of BD CSampler Software with the exception of instrument control functions Thresholds and compensation can still be set BD C6Sampler Analysis Software is not intended for operating the flow cytometer MINIMUM SYSTEM REQUIREMENTS e PC version Intel Core Duo processor 2 8GHz 2GB RAM CD ROM Drive Windows XP Service Pack 2 Windows Vista or Windows 7 recommended Language selection US or UK English 5GB hard disk space Display resolution 1280 x 1024 or higher e Mac version Intel or PowerPC processor 1 66 GHz Intel processor recommended 1GB RAM CD ROM Drive Mac OS X software Language selection US or UK English 5GB hard disk space Display resolution 1152 x 864 or higher 1280 x 1024 recommended SOFTWARE OPERATION See the relevant chapters in this manual for operational i
94. er Software does not automatically save the file Save these changes manually see section 4 12 2 To enable or disable auto save e Select File gt Auto save Settings e Doone of the following in the Auto save Settings dialog box e Select the Auto save Enabled radio button to enable auto save e Select the Auto save Disabled radio button to disable auto save Auto save Settings The application automatically saves sample data after it is acquired Auto save will occur at the completion of data acquisition in each well Auto save will only save the most recent data collected Auto save Disabled OK Figure 4 41 Auto Save Settings Dialog Box e Click on the OK button to accept the change and close the dialog box e f prompted to save the workspace before closing do one of the following e Click onthe Yes button to save the entire workspace e Click on the No button to exit the dialog box without saving the workspace 7820099 01 Rev 0 51 BD CSampler Software User Guide 4 13 52 4 12 2 Manually Saving Files To manually save a BD CSampler Software file e Select File gt Save To manually save a BD CSampler Software file with a new name e Select File gt Save File As Save 0 a fees laoo Save In J BD Accuri CSampler ve 6 8 023 BD D File Name SAMPLER ALIGN TEST TUNE 20120402 Files of Type Workspace c6 Figure 4 42 Sav
95. ess 71 5 6 2 Selmgth Threshold wsecceesasusncctienssensentseendteuedeaieatsuastiasatengeskseensieuategzantdeesiuss 72 5 6 3 Setting a RUN Uni eatecerctscoectresoreeccivncttieyenee idol A AEN 72 504 ANGSTEN 72 5 6 5 Washing the SIP Between Samples rrrnrrnnnnrvnrnrrnnnnnennnrrnrnnrennnnrennerennnernnnn 73 5 6 6 Changing Run Direction ccccccecccseccceeeceececeecceeeceueeseeeseueesseeeseeessueenaess 73 5 7 Creating the Sample Annotation Table rrrrnnrrnnnnrrnnnnrnnnnrnnnnrrnnnnrrnnnrennnrrnnnnrennnnennnn 74 5 6 USING INE FUN DIS DIAY sara 75 5 8 1 Acquiring Data in the Auto Collect Tab rrrnnrrnnnnnnnnnnrnnnrrnnnnrrnnnnrnvnnrennnnrenn 75 582 SPNO Data COEN ersan 76 5 8 3 WICWING SaAmple PS suv see 77 5 9 Ending a Data Collection Session rrrrararnannerrarrrrnnnenranenrnnnennnnrrnnnnennanenrnnsennnnennnn 78 5 10 Saving a BD CSampler Software File rrrrnnrrrrnrrrrnnrerranernnnnernnnrnnnnnennnnennnnnennnnennnn 78 5 11 Creating a BD CSampler Software Template rrrrnrrrrnnrrnnnnrrnnnnrrnnnnrnnnrrnnnrrennnrrennn 78 g2 EXDOMING APG IMPOMING FICS crsa a adr dei neiddubenderenanduds 79 518 EXPONO Sample SEWN He 79 ANALYZING SAMPLE DAT Aage 80 6 1 VIEWING ING Analyze TaD suasaruavvsvravasesvvatavvunthvagannventluvataanendt add tha 80 62 Sep PR sr 82 6 2 1 Copying Plots from the Manual Collect Tab rarnnnnnnnnnnnnnnnnnrnnnnrnvnnnrnnnnrnnnn 82 622 Creating PiSa4a4rrd ST 83 6
96. etails details See section 8 2 1 for details samples See section 4 2 8 for details See section 4 15 for details Set Threshold Sets the event threshold to gate out debris and noise from ene cell samples The default value is 80 000 on FSC H See section 4 2 2 for details on setting threshold values RUN PAUSE ADD TO Toggle button that performs the following functions e RUN Starts the sample acquisition e PAUSE Pauses the acquisition Click on ADD TOto resume data collection e ADD TO Allows additional sample collection into a well that already contains data Set Color Compensation Opens the Color Compensation dialog box for correcting fluorescence spillover See section 4 14 for details Acquisition Counters Displays the following information about the most recent acquisition for the selected well Last Run and all acquisitions for the selected well Cumulative in real time Events Number of events sampled Time Elapsed acquisition time Microliters Volume of acquired sample Events sec Events acquired per second When the run is completed this is the average value Events uL Events acquired per microliter When the run is completed this is the average value 24 7820099 01 Rev 0 BD CSampler Software User Guide 4 2 Delete Events Permanently deletes all events from the current sample Enable the Show warning check box to display a warning message before deleting sample data Also contains a Data
97. first empty data well e Select File gt Import FCS File e Navigate to the location of the file Open Look In a 20110413 154400 cos cca Be A01 unstcs A02 CD45F fcs Y A03 CD8 PE fcs 7 A04 CD56 16 PECy7 fcs Y A06 45 8 3 4 fcs Y B01 45 8 56 16 4 fcs B02 45 8 56 16 3 4 19 fcs C02 fcs Y B03 3 8 4 19 fcs B04 3 8 56_16 4 fcs A05 45 FITC 3 PCy5 19 APC fcs a B05 3 56_16 19 fcs a B06 water fcs Y c01 fcs Y c03 fcs c04 fcs Y c05 fcs Y CO6 fcs gt File Name lA01 unstfcs Files of Type FCS fcs x Open Cancel Figure 4 55 Open an FCS File 62 7820099 01 Rev 0 BD CSampler Software User Guide e Select the file and click on the Open button e Multiple FCS files from within a folder can be selected and imported as a group using shift click Files begin importing into the selected data well and proceed in succession horizontally from left to right wrapping to the following row once the current row is filled NOTE Only FCS files created by BD CSampler Software can be imported into a BD CSampler Software file or template 7820099 01 Rev 0 63 BD CSampler Software User Guide 5 AUTOMATIC DATA ACQUISITION The Auto Collect tab allows automated sampling from a well plate or sample tube rack The tab contains buttons and controls for perform
98. fter a specified number of events have been sampled e Enable the Run with Limits check box e Enable the check box next to the events field e Inthe associated text box type the number of events at which to stop the run e Doone of the following in the drop down list below the text box e Select Ungated Sample e Select a gating strategy if one exists to stop the run when the assigned number of events has been collected in the gated region To stop the run after a time has expired e Enable the Run with Limits check box e Enable the check box next to the Min and Sec fields e Type the number of minutes Min and seconds Sec at which to stop the run To stop the run after a specified volume has been sampled e Enable the Run with Limits box e Enable the check box next to the ul field e Type the volume in microliters uL at which to stop the run 7820099 01 Rev 0 31 BD CSampler Software User Guide 32 4 2 7 Running the Sample Gently resuspend the cells in the sample tube or plate and load the tube rack or plate Select a well in the sample grid that corresponds to the sample location on the plate or tube rack Click on the RUN button to start the sample collection BD CSampler Software begins fluidics initialization During this time the Traffic Light turns yellow and the software displays the message Preparing to analyze sample Once initialization is complete the Traffic Light turns green and the s
99. gion renaming e Event coloring in a region e Vector scalable graphics e Batch analysis NOTE Enhanced Analysis is compatible with BD Accuri C6 Software and BD CSampler Software version 264 and higher F 1 Creating a Live Gate Live gating also called dump gating is used to exclude events outside a designated region from being displayed and stored as part of the c6 or fcs file Any region may be used as a live gate This selection can be remembered as part of the template if desired CAUTION Once a live gate is executed there is no way of recapturing any excluded data To create a live gate e Create the region to use in the live gate in the Collect tab e Click on the Do not collect events outside check box and select the region from the drop down list located under Run Settings in the Collect tab E 10000 events in ungated Sample jo Min 0 Sec o pl h Do not collect events outside R1 Y Backflush _Unclog Figure F 4 Creating a Live Gate 7820099 01 Rev 0 127 BD CSampler Software User Guide F 2 Renaming Plots and Regions Plots or regions in a plot can be renamed In each case the method is the same To rename a plot or region e Double click on the plot or region name Plot 1 LATEST H0 way Figure F 5 Enable Renaming of the Plot e Inthe text field type in anew name Hew Hamel Ieper Jald Figure F 6 Type a New Plot Name e Press Enter New Name 405 RBC 4 t1 GATE No
100. h sample is complete It is also possible to manually save data at any time To manually save sample settings e Click on the Apply Settings button The first time settings are applied BD CSampler Software prompts the user to name the file and designate a file location 5 5 5 Removing Settings To remove settings from a sample set e Doone of the following e Select one or more wells in the set e Double click on a well in the set to select the entire set e Click on the Remove Settings button Each selected sample well reverts to a white box and is removed from the set NOTE Acquisition settings displayed in the Control Panel do not change Defining Data Acquisition Settings Data acquisition settings must be defined to create a sample set for an auto run This section gives details on assigning the following settings e Fluidics rate e Threshold e Run limits e Agitate cycles optional e Wash cycles optional e Run direction 5 6 1 Setting the Fluidics Rate The flow cytometer can accommodate an upper limit of 10 000 events per second but it is recommended to acquire samples at a rate of 2 500 events per second or less to ensure the best data resolution NOTE If fluidics settings were modified in the Manual Collect tab they do not carry over in the Auto Collect tab To set the fluidics rate 7820099 01 Rev 0 71 BD CSampler Software User Guide 72 e Click on the Slow Medium or Fast radio button in the
101. hat mimics voltage and amp gain adjustments to reposition data on the axis after the data has been collected VirtualGain makes gross adjustments approximate visual shifts of the data of histogram plots It is strictly an analysis tool and should not be used while collecting data For example in Figure 9 1 the negative peaks in the control sample and in sample 1 fall in similar channels mean value 28 2 and 29 7 respectively However the negative population in sample 2 is farther to the right mean value 73 4 VirtualGain can be used to align the negative peak of sample 2 with the control sample Control Sample Sample 1 Sample 2 10 000 10 000 10 000 Negative peak Count 5 O00 Count 4 O00 a FL1 A Overlay of Control Sample1 and Sample 2 10 000 10 000 E control 2 E Sample1 HH sample ee O T ane FL1 A FL1 A Original Data VirtualGain Applied to Data Figure 9 1 Before and After Applying VirtualGain 7820099 01 Rev 0 BD CSampler Software User Guide 9 1 Applying VirtualGain Only apply VirtualGain on a histogram plot and only on one parameter at a time After VirtualGain is applied data can be viewed in any type of plot and VirtualGain can be toggled on and off VirtualGain is only applied to the displayed data and does not alter FCS data The adjustment is recorded only in the BD CSampler Software file To apply VirtualGain In the Analyze tab do one of the following e Recr
102. he pop up dialog box for information about gates see section 4 7 Plot 2 401 8 peak GATE RA in all Gate applied Figure 3 11 Gate Applied to 8 Peak Bead Plot 7820099 01 Rev 0 BD CSampler Software User Guide Measure the CV of the top brightest far right peak on each of the three fluorescence plots Do the following to place the pre drawn horizontal marker tightly around the peaks e Use the Zoom Tool amp in the plot to zoom in on the top peak see section 4 11 e Adjust the marker tightly around the peak by clicking on the marker and dragging its edges e Click on the Expand Tool to zoom back out Plot 2 A01 amp peak GATE R1 in all Figure 3 12 Zoomed View of Plot Compare the bead run to the manufacturing results sent with the flow cytometer If the flow cytometer is performing properly the data plots should look similar to the 8 peak bead plots in Figure 3 13 Look for the following e One main population of beads on FSC H vs SSC H a shadow population is acceptable e Eight discernable peaks on FL1 H and FL2 H e Atleast six peaks on FL3 e Top peak CVs are less than 5 NOTE FL4 H performance is validated using the 6 peak beads described in steps 6 9 7820099 01 Rev 0 17 B 18 D CSampler Software User Guide Plot 1 404 8 peak 2654 Plot 2 A01 8 peak 2654 kA Plot 3 A01 amp peak 2654 x GATE No Gating GATE R1 in all GATE R1 in all Plot 4 A01 8 peak 2651
103. ics tab is organized into two major sections e Setup controls for the Master Statistics Table e Master Statistics Table When the Statistics tab is first opened the workspace is empty To create a Master Statistics Table select data from the setup controls BD CSampler BD Statistics Display Statistics Column Selector Plot Preview AO ae a ee et eee acel eoume ok Peer of Ths Plat ET Mean kse NU Mey H cesen ha Riedy Mer aseri os B E S E c a E D T e S E a E D a E E O E E E E E a E E a S E a E Hetet tars o feen ep uns sagnant oe eb Hup ee Sas a a a SS n E ae E a a E EE E E S Plot 3 FL1 H Count volume yt Events ut of This Plot of AF MeanFLIH CVAIH Medani H 3 gt ae E o ot 1 No Sample Gate No Gating pi Plot I FSC H SSC H _ Plot 1C FSC HISSC H Plot 3 FL1 H Plot 3C FLI H Plot 4 FL2 H BE TOLL ET SE A o ET ri Plot 4C FL2 H gt gt VG VE Plot 5 FL3 H oe _ _ _ fF ONE ETE V OE EDI EE TE Plot SC FL3 H nn I tt Plot 7 FSC HISSCH ium SE 2 gt ONE gt Vs FG DE GE gt es VNR a VE gt 6 ee ee GE a SLSR p p 73 2 H Count Vad ima fed Vl Fuerte lid 195 af The Plat 195 of AB Mean F DH 1 CV RO H Median FI DH ample Select Master Statistics Table Add rows to your master statistics tab by wale Select cells from the Sample Selector and Statistics Column Selector to build your table Add
104. ing the following functions Loading and ejecting a plate Creating sets of samples with the same acquisition settings Acquiring data Setting stop criteria and thresholds Controlling the fluidics Optionally washing the SIP between samples Optionally agitating mixing samples Viewing sample information 5 1 Viewing the Auto Collect Tab The Auto Collect tab is organized into two major sections Instrument Control Panel Panel on the left side of the window that contains controls for collecting data Sample Annotation Table Large table on the right side of the window used to name samples rename parameters and add a notation to each sample BD CSampler Auto Collect Plate Type 24 tube rack ge Sample Name Rename FL1 Rename FL2 Rename FL3 Rename FL4 Se TE pricier TT Plate Name gt 1 t 1 1 14 Ctrl ve to view rs se eT DeselectAll p Y Bpk verification run Bpk Verification 10m run Bpk Verification 10m run 10m run TETTE ene VE ooo oo o CF3 Fa FS Fo F7 Fe Fo FIO Fit FIZ peses JE 03 64 65 06 67 08 69 610 611 612 H1 H2 H3 H4 HS He H7 HB H H10 H11 H12 C6 is connected and ready Fluidics V 10000 events Slow Medium Fast S es Flow Rate 14 pL min in Ungated Sample Y Core Size 10 um lo Min 0 FP w Set Threshold Delete events on Do not collect events outside R1 less than fo Wash Settings None Agitate Plate None evey fi Well s None every
105. kinked fluidic lines 7820099 01 Rev 0 93 BD CSampler Software User Guide 94 2 3 Run a decontamination cycle from the Instrument Menu or by restarting the cytometer Within 5 minutes of completion of the decontamination cycle place a 12x75 mm tube containing 750 uL of 70 ethanol on the SIP Acquire 400 uL on Fast fluidics setting Within 5 minutes of completion of the ethanol run place a 12x75 mm tube containing 1500 uL of filtered deionized water on the SIP Acquire 400 uL on Fast fluidics setting Within 5 minutes of completion of the water run place a calibration sample on the SIP Select Instrument gt Calibrate Fluidics e Calibration should be performed in the same tube as the experimental sample e Calibration should be performed using a sample of the same or similar viscosity as the samples to be analyzed For example if lysed human peripheral blood samples are to be acquired lysed human peripheral blood should be used during calibration e The volume in the control sample tube should be 110 ul more than the volume used with subsequent test samples For example if using 1000 ul samples perform calibration with 1110 uL in the tube The calibration procedure consumes approximately 220 uL The values determined by the BD Accuri C6 are based on the average sample height in the tube during the calibration e f sample volumes gt 50 uL are to be acquired from the sample tube the calibration volume shoul
106. l possible solutions e Increase the primary threshold channel taking care that the increase does not remove cells of interest from the data set e Include a secondary threshold taking care not to exclude cells of interest e Dilute the sample Fluidics rate settings and sample core size can be adjusted to accommodate very small or very large particles For information on customizing these settings see APPENDIX B 4 2 2 Setting the Threshold Use thresholds to gate out light scatter and or fluorescence signals caused by debris in cell samples and electronic noise inherent in the system When set correctly for any given sample set resolution of particle or cell light scatter and fluorescence signals is greatly improved and data set size often can be reduced By default BD CSampler Software is set to a primary threshold of channel 80 000 on FSC H Notes on setting thresholds Threshold settings can be changed before during or after data acquisition but the most consistent predictable results will be obtained if threshold settings are chosen before final data collection for any given experiment The primary threshold is the parameter that triggers data collection Optional secondary thresholds can be applied to filter out additional data 26 7820099 01 Rev 0 BD CSampler Software User Guide All thresholds are set on the Height signal for any given parameter For best results when setting or changing thresholds create a plot th
107. lick on one of the following icons in an empty plot corral Density Plot Dot Plot 21 Histogram Plot ss 7820099 01 Rev 0 35 BD CSampler Software User Guide BD CSampler Software displays an FSC A vs SSC A plot by default Flot 1 4017 Unstained Flot 2 404 Unstained x GATE No Gating GATE No Gating en 10 000 12 800 oo oO di Sl ms D 500 000 1 000 000 1 600 000 0 5 000 000 10 000 000 16 777 218 FSC A FSC A Soe m A EL see H Figure 4 13 New Density and Histogram Plots e Configure plot specifications as needed see section 4 5 for details 4 5 Changing Plot Specifications The Plot Spec Tool allows manipulation of the data display in a plot including axis parameter selection channel range specification and selection of linear or logarithmic axis scale The Plot Spec Tool is available in the Manual Collect and Analyze tabs Set up or modify plot specifications at any time before or after collecting data To change the plot specifications e Click on the Plot Spec Tool icon Min Value Max Value C lag Hide 1st 0 1 600 000 Iw eee Range 0 16 777 215 AXIS 550 YF linear Min Value Max value f log Hide 1st U 800 000 e PE Range 0 16 777 215 Apply DE Cancel Figure 4 14 Set Plot Specs Dialog Box 36 7820099 01 Rev 0 BD CSampler Software User Guide e Inthe Set Plot Specs dialog
108. lied to the entire plate while acquisition parameters wash settings and run limits can be applied to individual sample sets Define the file name for auto save Create the Sample Annotation Table Select the direction to run the plate horizontal or vertical Open the Run Display Select the plots to view during acquisition Plots created using the manual collect tab are available for viewing Click on the AUTORUN button When the plate is complete close the Run Display Assigning a Plate Type When setting up an experiment select the appropriate plate type NOTE Changing to a new plate type results in BD CSampler Software opening a new blank workspace To assign a plate type Select one of the following options from the Plate Type drop down list e 96 well plate U bottom e 96 well plate flat bottom e 96 well plate V bottom e 96 deep well plate e 48 well plate e 24 tube rack 7820099 01 Rev 0 BD CSampler Software User Guide Auto Collect Plate Type hl Plate Name 96 well plate U bottom 96 well plate flat bottom 96 well plate V bottom Select All Cir click to vilgg deep well plate DeselectAll 245 well plate 24 tube rack Selecta Plate Type Figure 5 2 Plate Type Drop Down Menu The sample grid displays the available wells for data collection based on the plate assignment 96 well plate assignment Displays all 96 wells 48 well plate assignment Displays rows A F columns 1 8
109. lied with the Selectable Lasers software See section E 4 for details E 2 Validating Proper Function After Installation After installing the Selectable Lasers Module verify that the module is operating properly To verify the Selectable Lasers function 116 Leave the standard optical filters in place With BD CSampler Software open and the flow cytometer powered on select the 3 blue 1 red laser configuration 2 blue 2 red 4 blue Figure E 6 Selectable Laser Options 3 Blue 1 Red Set the run limit to 30 000 events in all and set the fluidics rate to slow Collect data files for the 8 and 6 Peak Validation Beads PN 653144 and PN 653145 Confirm that the flow cytometer is operating within specification see Chapter 3 7820099 01 Rev 0 BD CSampler Software User Guide 3 blue 1 red 2 blue 2 red Select the 2 blue 2 red option and collect data using the 6 Peak Validation Beads PN 653145 into a new data well 3 blue 1 red Figure E 7 Selectable Laser Options 2 Blue 2 Red Select the 4 blue option and collect data using the 8 Peak Validation Beads PN 653144 into a new data well 3 blue 1 red 2 blue 2 red Figure E 8 Selectable Laser Options 4 Blue Confirm proper operation of the 2 blue 2 red configuration by comparing the 6 peak bead distributions for FL3 and FL4 to those obtained with the standard 3 blue 1 red configuration Results for the 2 blue 2 red selection should show 6
110. lly selected for printing This box can be de selected if desired e Select File gt Print Selected Items 7820099 01 Rev 0 61 BD CSampler Software User Guide NOTE Printing directly from BD CSampler Software will result in low resolution images even if the eps option is selected in the Set Plot Drag and Drop Format dialog box 4 19 Exporting and Importing Files Data can be exported from sample wells from the Manual Collect or Analyze tab as individual FCS 3 0 files at any time NOTE See Appendix F 4 for information about saving plots as publication quality images To export data e Doone of the following e Select File gt Export FCS File to export and save the currently selected data well as an FCS 3 0 file e Select File gt Export ALL Samples as FCS to export and save all data wells as individual FCS 3 0 files e Select File gt Export ALL Samples to Third Party to export and save all data wells as individual FCS 3 0 files that enable autoscaling in third party applications such as FlowJo e Select File gt Export Plot Data as CSV to save an individual file in csv format see APPENDIX H for an example csv file e f prompted to confirm the export click on the OK button To import an FCS data file into BD CSampler Software e Select an empty data well ina BD CSampler Software file or template If the currently selected data well contains data BD CSampler Software will start FCS file import in the
111. lot e Select File gt Set Plot Drag and Drop Format e Select the eps option in the dialog box e Click on the OK button e Inthe BD CSampler Workspace click on a plot and drag it onto the desktop e Open an image editing application e g Photoshop and ensure the image resolution is set to 300 dpi or higher if required and import the eps image e Save the image F 5 Analyzing Batches of Samples 130 The Batch Analysis tab allows an automated analysis to be performed on multiple samples at the same time The plot types created during acquisition will appear at the top of the screen Statistics can be displayed for individual files or all files selected After analysis regions can be moved or resized on plots for individual samples without affecting the plots for other samples F 5 1 Viewing the Batch Analysis Tab The Batch Analysis tab is organized into two major sections e Setup panel Panel on the top half of the window that contains controls for selecting samples and plots for analysis 7820099 01 Rev 0 BD CSampler Software User Guide e Data display Large area on the bottom half of the window that shows sample data in plots and in a Statistics Table When the Batch Analysis tab is opened for the first time the workspace is empty To set up the Batch Analysis tab plots can be created or copied from the Manual Collect or Analyze tabs Batch Analysis VW Show Plots from Manual Collect Show Plots from Ana
112. lyze SelectAll DeselectAll tented madd Pict t Mot 2 Plot 3 Pot 4 Plot Pot 9 ro AP AB AG AIO Alt AIS a Gate No Gating Gate No Gating g Gate RI S Gate Rt B Gate R1 except N Gate No Gating JED E GEE DER ER ee ha I n p S T 4 Ba es os BP E aa Bi BAR mi 1 c2 cal ca cs co e G8 Go Eto Git G T fe 5 26 z I y 4 R 5 gt 59 100 0 Ss 5 r R3 d D o 02 03 04 os ps BP ba w BOB Be a 100 0 98 Bes y 28 28 ma D amp 00 0 i n smed wo HP Mt Oo N M3 er amp Pl Oo o i o o 4 A Fer lt jaeeaser ss EWEN en joo Nidan oa Mom Hideo PS MAS PES S ben n N A i VEKE SG a gt r r o Hr r a nes a I i o i aa p 4 Fite F LEJ Fa fs P Kil ra oO mo ra m2 r 9 t S p 510 202 000 000 n 407 pc w par prp 2 Rie pee pg pr dr pre 2 kg 7 s hp pep pep br 2 SAA j Hh ON OG OG 04 OG OG 67 OG Ga Sid Smi ore EE J se GE isa JL sa L ans JL Vi HMM hiia I f f I Show Statistics with all Plots Figure F 11 Batch Analysis Tab Workspace The following table describes each of the controls and indicators in the Batch Analysis tab Table F 1 Batch Analysis Tab Controls Sample Grid Matrix laid out in the configuration of a multi well plate for selecting the samples to analyze The wells are color coded White Does not contain data Blue Contains data Black check mark Curre
113. m value under Y AXIS e Click on the Apply button to apply the changes and click on the OK button to close the Plot Spec dialog box 4 12 Saving a BD CSampler Software File A BD CSampler Software file is a comprehensive and often large data file that contains instrument settings FCS files and plot layouts The file contains the entire workspace including the following elements Sample data Plot layouts Zoom levels Gating Color compensation Threshold settings Collect tab settings Changes made in the Analysis or Statistics tabs 50 7820099 01 Rev 0 BD CSampler Software User Guide By default BD CSampler software automatically saves data at the end of each sample run Data can also be saved manually To save the entire BD CSampler file save the file manually See section 4 12 2 When a file is saved the software displays the file name in the upper left corner of the workspace Figure 4 40 bd BD CSampler software file File Edit Display Instrument About name BD CSampler Figure 4 40 Title Bar with File Name 4 12 1 Auto Saving Files By default BD CSampler Software automatically saves the event data any time the flow cytometer reaches a run limit or if a run is paused Auto save does not save changes to acquisition settings plots or gating strategies that occur after the initial save when naming the BD CSampler Software file CAUTION If changes are made after a run is paused or completed BD CSampl
114. mal 7820099 01 Rev 0 BD CSampler Software User Guide 8 6 Technical Support For Technical Support contact In the USA Accuri Cytometers Inc 173 Parkland Plaza Ann Arbor MI 48103 USA Phone 1 734 994 8000 Fax 1 734 994 8002 Customer Support CustomerService AccuriCytometers com Technical Support TechSupport AccuriCytometers com e Europe Middle East and Africa BD Biosciences Erembodegem Dorp 86 9320 Erembodegem Belgium Phone 32 53 720 882 Customer Support bd accuri europe bd com Technical Support techsupport europe bd com e Canada BD Biosciences 2100 Derry Rd West Suite 100 Mississauga Ontario Canada L5N 0B3 Email canada bd com Direct Phone Numbers Austria 0810 101 807 Belgium 078 166 050 Canada 1 800 268 5430 Denmark 8025 0622 Finland 0800 915582 France 0811 290 069 Germany 0180 100 1732 Ireland 1850 930 396 ltaly 840 999 926 Netherlands 0900 0400 142 Norway 800 17 382 Poland 00800 121 4744 7820099 01 Rev 0 95 BD CSampler Software User Guide 9 ADJUSTING PEAK POSITION WITH 96 VIRTUALGAIN In certain instances a particular peak should have the same position across different samples or be located at a specific channel number regardless of the staining Instruments that have voltage and amp gain controls allow peak position adjustments from sample to sample BD CSampler Software uses VirtualGain instead of these controls VirtualGain is a software module t
115. measurement Update Firmware Updates the flow cytometer firmware Use only when directed by BD Accuri to upgrade the firmware with an official firmware release 7820099 01 Rev 0 10 Q BD CSampler Software User Guide _ ne P feature that allows control of the flow P from a remote location E About BD CSampler Software Opens a dialog box that displays the version of BD CSampler Software and Accuri Technical Support contact information Technical Support Opens a dialog box that displays information Information about BD CSampler Software and the BD Accuri C6 flow cytometer Each time an activation key is used to install a new BD CSampler Software component the dialog box is updated to reflect the change Users Allows addition deletion or modification of user accounts with the optional user tracking feature 104 7820099 01 Rev 0 BD CSampler Software User Guide APPENDIX B ADVANCED FLUIDICS SETTINGS In the Manual mode advanced users can customize the fluidics rate and core size for collecting samples To customize the fluidics rate e Select the Custom radio button in the Fluidics section of the Manual Collect tab Fluidics f Stow C medium Fast f Custom Flow Rate pL min Sel Core Size Core Size ym Threshold Set Threshold 20 000 on FSC H Hone Figure B 1 Custom Radio Button e Move the Custom slider to adjust the flow rate To customize the sample co
116. mn 1 12 or row header labels A H to select the entire column or row Multiple rows and columns can be selected simultaneously e Click on Select All above the Sample Grid to select all the wells Selected wells contain a black check mark 7820099 01 Rev 0 BD CSampler Software User Guide Aulo Collect Select All i t r CFS IS IIA d OF Oe OG 090 O11 OT Sc cei ce C90C9111C42 5 Of 08 06 040 041 pn Ea 9 t0 EIT Et 7 FO FO rotna G7 OG GF 640641 617 o Kl MG HIO WIT HU e 3 bive 1 ree connected and ready 2 bl t 2 red dhna Figure 5 5 Selected Wells in the Auto Collect Tab e Enter settings for the following Fluidics see section 5 6 1 Threshold see section 5 6 2 default is FSC H 80 000 Run limits See section 5 6 3 Agitate cycles see section 5 6 4 optional Wash cycles see section 5 6 5 optional NOTE If run limit or fluidics settings were modified in the Manual Collect tab those settings do not carry over in the Auto Collect tab Threshold settings and color compensation settings do carry over e Click on the Apply Settings button The selected samples are highlighted in the same color 7820099 01 Rev 0 69 BD CSampler Software User Guide 70 Auto Collect Plate Type 96 Jeep well plate v Plate Name Select All Cirt lt ick to view sample setings Deselect All Bt 82 8 amp DS OO br BP BO MIO Btt O12 ct c2 c3 gt 04 C5 C8 Cc OG CO C10 Ctt C1
117. mple well type in the following information Click on each cell and type appropriate information Renaming parameters in the Sample Annotation Table will be also applied to data plots To copy and paste information from a spreadsheet e Highlight fields in the spreadsheet e Press Ctrl C e Inthe Sample Annotation Table click on Sample Name of well A1 or other appropriate cell e Press Ctrl V to populate the Sample Annotation Table with the highlighted data from the spreadsheet 7820099 01 Rev 0 BD CSampler Software User Guide 5 8 Using the Run Display The Run Display is used to begin view interrupt and abort data collections in Auto Collect The Run Display contains the following elements e Status banner e Two large plots e Data acquisition counters e Two pause options To open the Run Display e Apply settings to one or more wells See section 5 5 1 e Click on the OPEN RUN DISPLAY button The Run Display opens in the main panel of the Auto Collect tab BD CSampler Figure 5 11 BD CSampler Run Display To close the Run Display and return to the Control Panel e Click onthe CLOSE RUN DISPLAY button 5 8 1 Acquiring Data in the Auto Collect Tab BD CSampler begins data collection in the first well that has settings applied most commonly A1 and progresses horizontally or vertically as defined in the settings As data is acquired and BD CSampler advances thro
118. n setting fluorescence compensation see section 4 14 4 11 1 Basic Zoom e Click on the Zoom Tool e Draw an area to zoom on by clicking and dragging the mouse in the plot Plot 1 Sample Unstained Plot 1 Sample Unstained GATE No Gating GATE No Gating Zoom 2 a a p a a a ea 2 a a sF 2 a 2 a a 0 200 000 485 715 FSC A JEPP FAK 0 00 000 1 000 000 1 600 000 FSC A OO mA Figure 4 38 Before and After Using Zoom Tool e Repeat steps 1 2 as needed to zoom in To zoom out e Click on the Expand Tool e Repeat step 1 as needed 7820099 01 Rev 0 49 BD CSampler Software User Guide 4 11 2 Zooming to a Specified Channel Range Sometimes it can be helpful to view a plot in a specified channel range To view a specified channel range in a plot e Click on the Plot Spec Tool Set Plot Specs for Plot 4 Ea AXIS CDSFL f linear Min value Max Value 4 log 100 1 000 000 pe Kiss tat Range 10 16 777 215 AAIS cod FY linear Min value Max Value fe log Hide 1st 10 100 000 v Range 10 16 777 215 Apply OK Cancel Figure 4 39 Plot Spec Dialog Box Set Min and Max Channel Values for the X and Y Axes e Specify the x axis channel range by typing a minimum Min Value and maximum Max Value value under X Axis in the Set Plot Specs dialog box e Specify the y axis channel range by typing a minimum and maximu
119. nal gate position in Manual Collect does not change e Click on a sample well to view data e Apply a gating strategy if desired see section 4 7 2 for details BD CSampler Plate Type 96 well plate U bottom v Plate Name A04 3 456 eo D7 DO DO B10 B11 DIZ DI 02 DD D4 DS DO 1 o2 f 03 cal ca f co or co co 2 0D p p D 2 J Bale 06 a7 ro ra re ro r7 ro eS WO OW WO We HIO MIT HZ 7 8 9 10 11 12 C10 011012 7 00 DO 01000110D42 ro rio ray riz 30 99 610011 0412 Make a new plot hal zi l bA Plot 2 p C A SSC A Plot 3 FLI A PLJ A Plot 4 FLI A PLAA AG AIO All A12 oo EO 10 11 142 Analyze Figure 6 8 Analyze Tab Plots with Gating Applied To view data from another sample open one or more plots from the Plot List it is recommended to do this in another row of plot corrals and choose the sample to be displayed in each plot Gating strategies applied above are automatically applied to the corresponding plots in the new row BD CSampler Plate Type 96 well plate U bottom bi Piate Name A02 HPB CD45 PE PDS SD V oE B2 B3 BA B5 c2 C3 C4 C5 D2 D3 D4 DS E2 E3 E4 E5 F2 F3 F4 FS G2 G3 G4 G5 66 sca 7m oa BS gt H2 H3 H4 HS H6 Copy Plots from Collect J Plot List Plot 2 FSC A SSC A Plot 3 FL1 A FL3 A Plot 4 FL1 A FL4 A 89 10 11 12 AT AB AD A10 A11 A12 B7 BS BY B10 B11 B12 C7 cs C9 C1
120. nennnnnennnnen 43 Figure 4 25 Parent Gate Applied to Dot Plot rrnnennnnrrnnnnennnnrnrnnnennnnrnrannennnnennnnnnnnnnennnnnennnnen 43 Figure 4 26 Second Gate for Creating Nested Gates rrrrnnnrrrnnnnrnnnrrnnnnnrnnnnrnnnnrennnnrnnnrrennnsen 44 Figure 4 27 Applying the Child Gate rrrrrrranrrnnnnerrannrnnnnenrnnrnnnnnennnnrnnnnnnnnnnennnnnennnnennnssennnsene 44 Figure 4 28 Third Plot with Nested Gate Applied R1 in P1 ranunnnnonnnnnnnnrnnnnnvnnrnnanennnnernnrnnnrr 45 Figure 4 29 Statistics of Plot with Nested Gate rrrarrnrnnrenranrrnnnnerranrnrannennnnenranrennnnennnnnnnnnnen 45 Figure 4 30 Selected Region in the Batch Analysis Tab ccccccccsecccceeceseeeeeseeeesaeeeeseeeesseeeas 45 Figure 4 31 Moved Region in the Batch Analysis Tab cccccccccsseccseeeceeeeseeesauseseeeseeesaueesaees 46 Figure 4 32 Before and After Changing Events Displayed cccscceceeeeseeeseeeeceeeeseeesaeeeneees 46 Figure 4 33 Events Display Settings Dialog BOx rrrarrrnnnnrnrarrrnnnnenrnnennnnnennnnennnnnennnnennnnnennnnen 47 Figure 4 34 Plot with Events Display Settings Applied rrrarrrrnnnernanrnrnnnernnnenrannnnnnnennnnnnnnnnen 47 FIGUe 435 PS LAD Peer ne nee ae ee ee a ee ee ee ee eee 48 Figure 4 36 Rename Parameters Dialog BOX ccccsecccseeeceeeeseeeseeeseeeeseeeseueesueeseeesaeeenaees 48 Figure 4 37 Rename Parameters Dialog Box with Axis Label Drop Down List 08 49
121. nformation 112 7820099 01 Rev 0 BD CSampler Software User Guide APPENDIX E SELECTABLE LASERS The detectors and lasers of the BD Accuri C6 flow cytometer operate in a predefined configuration detectors FL1 FL2 and FL3 read blue laser excited fluorescence emissions and detector FL4 reads red laser excited emissions This configuration is referred to as 3 blue 1 red The Selectable Lasers Module PN 653126 allows operation of the flow cytometer in two alternate configurations which significantly expands the fluorochrome combinations that can be analyzed see section E 4 for examples e 2 blue 2 red FL1 and FL2 read blue laser excited emissions FL3 and FL4 read red laser excited emissions e 4 blue All 4 detectors read blue laser excited emissions Components Supplied e Selectable Lasers Activation Key software e Three optical filters e 780 60 BP PN 653187 e 610 20 BP PN 653186 e 630 30 BP only available with Selectable Lasers Module NOTE The Selectable Lasers upgrade requires prior installation of BD Accuri C6 Software or BD CSampler Software E 1 Installing the Selectable Lasers Module e Verify that BD Accuri C6 Software or BD CSampler Software version 227 4 or above is loaded on the computer The Selectable Lasers Module can also be installed on any computer where BD Accuri C6 Analysis Software version 227 4 or higher is installed e Copy the Selectable Lasers Activation Key from the installation CD or flash
122. nt markers in the list of gates enable the associated check box es in the Change Gating dialog box Change Gating for Plot 4 Gating choices Include Click to view markers E Pl FSc 4 55C 4 8 Pl on all events FSC AJSSC Al Click on a gating option Clear All Figure 4 21 Selecting a Gating Option e Select one of the following gating icons e Include icon to analyze the events within the region Multiple gates can be included in a single plot e Exclude icon to analyze the events outside of the region e Intersection icon to analyze the events within the intersection of two or more regions 7820099 01 Rev 0 41 BD CSampler Software User Guide e Click on the Apply button BD CSampler Software displays the type of gate that is applied next to the GATE button in the plot Plot 2 Sample Unstained i GATE P1 in all Gate applied Figure 4 22 Plot Gated to Include P1 4 7 3 Creating and Applying Nested Gates A series of nested gates can be created in which each gate is a subset of the previous one Complex and informative gating strategies can be devised by the appropriate combination of nested gates using the Include Exclude and Intersection gating tools To create nested gates e Draw any region or marker around a population of events for example P1 Plot 4 204 HPB COS CO x GATE No Gating ae 200 O00 252 382 200 000 400 000 23 010 FSC A g0 mA of Figure 4 23
123. ntervals for up to 30 minutes If the interval is reached during acquire the BD CSampler will complete data collection prior to performing the agitation Agitation is only performed when the SIP is clear of any well or tube and does not interrupt data collection CAUTION Sample volume should not exceed 50 well capacity to effectively maintain suspension and avoid spillover with agitate For best results use U bottom 96 well plates or standard 12x75 mm tubes NOTE Agitate settings are applied to the entire plate or tube rack and cannot be applied to individual wells To set an agitate cycle e Select one of the following options e To agitate before a given number of wells click on the top radio button e To agitate after a given number of minutes click on the bottom radio button 7820099 01 Rev 0 BD CSampler Software User Guide e Select the number of agitate cycles Agitate Plate f Run Horizontally f None every hte NES f Run Vertically Figure 5 7 Agitate Plate Controls e Do one of the following e Type in the number of wells to increment between agitate cycles e Type in the number of minutes to increment between agitate cycles 5 6 5 Washing the SIP Between Samples Multiple wash cycles of the SIP can be specified in the Auto Collect tab Up to three wash cycles can be specified at a time One wash cycle typically reduces carryover to lt 1 0 Two wash cycles typically reduce carryover to l
124. ntly selected for batch analysis Available Plots Displays plots without data that can be included in the batch analysis Available plots include plots copied from the Collect tab or created in the Analyze tab See section F 5 2 for details Analysis Pane Rows of analysis data in table and plot format based on the plots that were selected for use in analysis Statistics Tables can be copied into most Microsoft Office compatible programs F 5 2 Running a Batch Analysis e Run a number of samples e After running the samples click on the Batch Analysis tab e Select the samples to analyze in the Sample Grid by clicking on the wells e Doone or both of the following e Enable the Show Plots from Collect check box to make all of the plots from the Collect tab available for batch analysis e Enable the Show Plots from Analyze check box to make all of the plots from the Analyze tab available for batch analysis 7820099 01 Rev 0 131 BD CSampler Software User Guide 132 Select the specific plots to include in the batch analysis by enabling the check boxes under those plots To show the statistics associated with samples do one of the following e Enable the Show Statistics with All Plots check box to show statistics in every row e Enable the Show Statistics check box in individual rows to view statistics for specific samples Regions can be moved and resized see section 1 F 5 3 Exporting Data Plots and
125. oftware displays the message Events are being recorded The well flashes blue during data collection After the run limit is reached the well tops flashing and remains blue indicating that the well contains data Additional data can be collected into a well that contains data at any time by clicking on the ADD TO button Note that run limits may need to be adjusted when adding data to a well BD CSampler BD Manual Collect Plate Type v Plate Name A01 Click Here To Rename Sample EL DIGG GT eS SRS Poe Bee oe ae A i 7 Plot 1 No Sample xX GATE No Gating SSC A 0 200 000 400 000 600 000 800 000 Select a Plate Type 0 500 000 1 000 000 1 600 000 FSC A ook A N eS NO a oe 2 2 2 2 2 5 12 2 Se Ea aaa Geta Gow feos KUN ie I Plot 4 No Sample 100 00 100 00 Last Run Cumulative 7 1 Events 0 00 0 Time 0000 All O Microliters o C Outside HE D Events Sec o pen of SE ron A Events Eee ae Figure 4 10 BD CSampler Software Workspace after Collecting Samples 7820099 01 Rev 0 BD CSampler Software User Guide 4 2 8 Washing the SIP Between Samples A wash cycle can be used to thoroughly clean the SIP between samples The flow cytometer aspirates the contents of the SIP up and out of the SIP then rinses the SIP and wash station with clean sheath fluid To wash the SIP Click on the Wash button in the Manual Collect tab 4
126. ometer Tighten with the installation and removal tool Figure 1 5 Secure the BD CSampler to the Flow Cytometer 7820099 01 Rev 0 BD CSampler Software User Guide CAUTION Do not completely tighten any bolt before all three bolts are partially screwed Completely tightening a bolt too early can cause shearing stress on the materials e Partially screw in the remaining two mounting bolts Figure 1 6 Secure the BD CSampler to the Flow Cytometer e Confirm that the module is lined up properly with the flow cytometer and completely tighten all three bolts e Connect the serial cable to the socket at the back of the flow cytometer Turn on the flow cytometer The BD CSampler automatically aligns itself and rests in the home position 4 7820099 01 Rev 0 BD CSampler Software User Guide Figure 1 7 Connect the BD CSampler Cable to the Flow Cytometer e Place the mat on the bench under the arm of the BD CSampler Figure 1 8 BD CSampler with Mat Installed CAUTION Failure to keep the mat clear of plates tubes or any other materials may result in damage to the BD CSampler Run validation beads to ensure correct BD CSampler operation see chapter 3 Validating the Performance of the BD Accuri C6 7820099 01 Rev 0 BD CSampler Software User Guide 2 BD CSAMPLER SOFTWARE OVERVIEW BD CSampler Software controls the BD Accuri C6 flow cytometer system with BD CSampler in order to acquire data generate stati
127. ometer with the solution in the flow cell allowing the flow cell to soak It is recommended to perform this cleaning cycle monthly or when a partial blockage of the SIP or flow cell is suspected To run the Extended Clean of the Flow Cell Cycle e Place a tube with at least 500 uL of Extended Cell Flow Clean cleaning solution PN 653159 NOT cleaning solution PN 653157 on the SIP CAUTION Never run the Extended Clean of the Flow Cell Cycle without a tube containing at least 500 uL of fluid e Select nstrument gt Extended clean of flow cell e After the flow cytometer is shut down leave the flow cytometer off for at least 30 minutes up to overnight for a more thorough cleaning e Restart the flow cytometer The flow cytometer performs a longer fluidics startup cycle and BD CSampler Software displays the message Extra startup time needed due to cleaning or improper shutdown This longer cycle purges cleaning solution from the flow cell and takes about 15 minutes to complete e Replace the tube containing cleaning solution with one containing 0 2 um filtered deionized water and run on Fast fluidics speed for at least 5 minutes to clear residual cleaner solution from the SIP and flow cell e Operate the flow cytometer as usual Cleaning the Fluidics Lines The Cleaning Fluid Cycle pulls cleaner fluid from the cleaner tank and runs it through the fluidic lines After filling the system with cleaner fluid the cleaning fluid c
128. ple C Program Files BD Accuri BD Accuri C6 Software ActivationKeys C Program Files BD Accuri BD CSampler ActivationKeys C Program Files BD Accuri BD CSampler Software ActivationKey frd Figure E 3 Install Wizard Install the Module e After installation click on the Close button Selectable Lasers Setup Installation Complete Setup was completed successfully Completed EEE EEL EEE ELLE EE ELL LLL Show details Nullsoft Install System v2 46 lt Back Close Cancel Figure E 4 Install Wizard Setup Completed Successfully 7820099 01 Rev 0 115 BD CSampler Software User Guide Open BD CSampler Software The Selectable Lasers controls are displayed next to the BD CSampler Software traffic light message A01 t2 5 4 5 6 T 0 910 i 12 AZ AS AF AB AG A AB AG A10 A11 A12 B1 B2 B3 B4 BSE BG BF BS BS B10 B11 B12 C1 C25C3 C45 C55 C6 I C7 FCS I C9 RC108C118C12 D1 D2 D3 D4 DS DG DF DS DS D10 B11 D12 E18 E2 ES I E49 ES f EG E7 I ES I E9 sE108 E118 E12 F1LEFZ AFS EFAS FOR FG R Fe FS FS EF108F11 F12 G1 G2 63 64 G5 G6 G7 G8 69 610611612 A B C D E F G H Hi HZ H3 H4 HS HG HF HS HA H10 H11 H12 3 blue 1 red C6 is connected and ready 2blue2 red C 4blue Figure E 5 Selectable Laser Controls in the BD CSampler Workspace Depending on the laser configuration to be used the standard optical filters may need to be replaced with one or more of the filters that are supp
129. ple The following figure shows the data after proper color compensation has been applied to the PE CY7 example in section 4 14 1 AOS HFB C045 PE Cy A05 HPB CD45 PE Cy AOS HPB CO45 PE Cy o Gate F3 in all Gate P3 in all k Gate P3 in alli mf LIL a mld 0 a E E E 3 Boo 3 a l gt T TG a En 3 Er w m H E H H g7 22 7 D 5 t OTs E at aot ADT HPB ADi HPB ADi HFB Gate P3 in all Gate F3 in all Gate P3 in all 8 DO0 3 000 6 000 6 000 4 000 4 000 Count 7 000 Partial data displayed Count 7 000 Partial data displayed Count Partial data displayed awl ata ah Ga ao t wl aot ae EE EE CD45 PE Cy7 A FL1 A A01 HPB Gate Mo Gating 4 000 45 000 Count O00 40000 175064 events displayed aol ae ae a aa an 2 FLAA Figure 4 45 Corrected Fluorescence Spillover 7820099 01 Rev 0 55 BD CSampler Software User Guide The PE CY7 fluorescence is now confined to the FL3 detector and no longer spills into FL2 or FL1 To correct fluorescence spillover 56 Click on the Quadrant Tool of a plot and click inside the plot Plot 2 A02 HPB CD45 PE Cy 7 GATE Pl in all T p D Ti ri F D Figure 4 46 Placing a Quadrant Tool Adjust the quadrant marker position so that all positive populations are cleanly contained in individual quadrants BD CSampler displays the median fluorescence channel value for the events in each q
130. ple Apply Close f All samples Figure 4 5 Threshold Settings Dialog Box e Click on the Apply button to apply the threshold settings e Click on the Close button to close the dialog box 4 2 3 Assigning a Plate Type e Select one of the following options from the Plate Type drop down list 28 7820099 01 Rev 0 BD CSampler Software User Guide 96 well plate U bottom 96 well plate flat bottom 96 well plate V bottom 96 deep well plate 48 well plate 24 tube rack Manual Collect Plate Type bd l Plate Name 96 well plate bottar 96 well plate flat bottom No Sample 96 well plate v bottom 96 deep well plate 3 10 11 12 48 well plate 24 tube rack j l Plate Type A B C D E F G H I T E m gmi Lap nn Ir Selecta 2 Figure 4 6 Plate Type Drop Down Menu The sample grid displays the available wells for data collection based on the assigned plate type 96 well plate assignment Displays all 96 wells 48 well plate assignment Displays wells rows A F columns 1 8 24 tube rack assignment Displays wells rows A D columns 1 6 NOTE Changing the plate type automatically opens a BD CSampler Software new blank workspace 7820099 01 Rev 0 29 BD CSampler Software User Guide 4 2 4 Naming the Plate e Name the plate by typing a label in the Plate Name field optional BD CSampler Manual Collect Plate Type Plate Name Click here to name plate i 2 3 4 5 6
131. pler Software an entry is made in the userUsage Log The userUsage log is a csv file Paste DP Format Painter Clipboard YONO U VN Page Layout Cut Calibri F 2011 Mar 25 22 25 09 2011 Mar 25 22 26 57 2011 Mar 25 22 27 25 2011 Mar 25 22 27 59 2011 Mar 25 22 28 46 2011 Mar 25 22 29 21 admin admin Clare R Clare R admin admin Figure C 8 userUsage Log Ama 2672 Sign in 2672 Sign out 2672 Sign in 2672 Sign out 2672 Sign in 2672 Sign out Wrap Text Alignment General fad Merge amp Center Af Technical Applications Technical Applications The userUsage Log contains the following information To view the userUsage log Date of a sign in sign out Time of a sign in sign out Username of the operator Serial number of the BD Accuri C6 Type of activity Sign in or sign out Navigate to the CytometerSupportFiles folder typically in the root directory of the BD CSampler Software computer and open the folder C Cytometer Support Files Share with Name New folder A __ commandHistory _ commandHistory log 2012 04 03 _ commandHistory log 2012 04 04 __ CytometerSwapFile exception _ LogLookupTable maintenance EL userUsage Date modified 4 5 2012 3 38 PM 4 3 2012 9 40 AM 4 4 2012 2 58 PM 4 5 2012 3 27 PM 4 3 2012 9 21 AM 4 3 2012 9 21 AM 4 5 2012 3 27 PM 4 3 2012 9 21 AM Figure C 9 CytometerSupportFiles Fold
132. ples select the Apply to All samples radio button in the Compensation Settings dialog box e Click onthe Save amp Close button to apply the color compensation settings 4 14 3 Troubleshooting Color Compensation Occasionally the plot might appear to have a smaller percentage of events in a quadrant than BD CSampler Software reports For example Figure 4 50 appears to have less than 26 1 of the population in the lower right quadrant even though the Statistics report 26 1 This occurs when a decade containing data is hidden In the following figure a number of events have been driven into channel 1 because of overcompensation These events are not displayed in the plot if the first decade is hidden but they are included in statistics calculations Plot 11 HPB C Chex 2257 pd GATE Pl in allj al s aot CE4 PE FL2 A Plot 11 HPB C Chex 2257 Count Volume pL of This Plot Gated on P1 in all This Plot 21 479 733 100 0 NT aul 7 73 0 0 CDa FITE FLAA Q1 UR 10 669 73 3 49 7 m GDF Quy 5 198 733 24 2 ger EN qR 5 605 733 26 1 Figure 4 50 Plot Displaying Overcompensation To fix overcompensation e Click on the Plot Spec Tool 58 7820099 01 Rev 0 BD CSampler Software User Guide e Inthe Set Plot Specs dialog box disable the Hide 1st decade check boxes for both X and Y axes Set Plot Specs for Plot 4 x ATS CDSFL a J linear Min value Max value fe log 1 16 777 215 go
133. ply settings and close the dialog box e Click on the Cancel button to close the dialog box without applying settings BD CSampler Software displays the plot with a message that some events are not being displayed Flot 1 404 Unstained x GATE No Gating 4 Events display settings applied 200 000 400 000 600 000 800 000 events displayed 25013 125064 20 1 sid d 1 000 000 1 600 000 OO fe Figure 4 34 Plot with Events Display Settings Applied Naming Plot Axes The axis labels can be renamed in any plot from the Manual Collect or Analyze tab to identify the antibody staining or fluorochrome used in the sample To name a plot axis e Click on an axis label and select Rename Parameters from the drop down menu Rename Parameters can also be accessed in the Edit drop down menu 7820099 01 Rev 0 47 BD CSampler Software User Guide Flot 1 201 Unstained x GATE No Gating ee Ea F 3 pa m So00 000 1 000 000 1 600 000 FSC A oOo X axis label Figure 4 35 X Axis Label e Inthe Rename Parameters dialog box type the new label in the edit box of the parameter Any parameter can be renamed from this dialog box Rename Parameters x Rename FL1 to ra finone Rename FL2 to a finone Rename FL3 to CD45 PE Cy7 FL finone a Rename FL4 to 4 finone Apply to Sample HPB CD45 PE Cy 7 OK aoe All samples
134. posed to light for long periods of time performance may be substandard Make new bead suspensions and run the bead sample again e There may be a bubble or clog in the flow cell Do one or more of the following o Run the bead sample again 7820099 01 Rev 0 BD CSampler Software User Guide 7820099 01 Rev 0 o In BD CSampler Software select the Unclog button and follow the software prompts When the cycle is finished a green Traffic Light will be displayed run the bead sample again o In BD CSampler Software select the Backflush button and follow the software prompts When the cycle is finished a green Traffic Light will be displayed run the bead sample again o Contact Accuri Technical Support 21 BD CSampler Software User Guide 4 MANUAL DATA ACQUISITION The Manual Collect tab is used to set the data collection criteria start and stop data acquisition and view data on collected samples The tab contains buttons and controls for performing the following functions Run individual samples from a plate or tube rack Settings such as thresholds and color compensation can be applied to either the current sample being viewed or to all the samples in the sample grid Set up experiments to be collected in the Auto Collect tab The following can be set up in the Manual Collect tab before switching to the Auto Collect tab 1 Create plots histogram dot or density for viewing data 2 Set up gating strategies 3 Set
135. r to improve data visualization or to normalize data sets This option allows visual removal of a number of events from the plot without deleting data Flot 4 003 HFE COS CD45 x Plot 4 003 HFE COS Cos x GATE No Gating GATE No Gating a 700 000 400 000 600 000 800 000 events displayed 700 000 400 000 600 000 800 000 JE267 1 231352 GOR 1 S00 000 1 000 000 1 600 000 600 000 1 000 000 1 600 000 FSC A apo ESKE ggo oz Figure 4 32 Before and After Changing Events Displayed 46 To change the number of events displayed in a plot Select Display gt Events Display Settings Do one of the following in the Events Display Settings dialog box To view all collected events select the Show all events radio button To view the first N events of a sample select the Display first radio button and type a number in the events collected field Use this option for data normalization To view a specified percentage of the whole in a pseudo random selection select the Display radio button and type a percentage to view for example if 20 is selected every fifth event is displayed 7820099 01 Rev 0 BD CSampler Software User Guide 4 10 C Display first 50000 events collected C Display 20 percent of events collected Apply Figure 4 33 Events Display Settings Dialog Box Do one of the following e Click on the Apply button to apply settings without closing the dialog box e Click on the OK button to ap
136. re displays the message Extra startup time needed due to cleaning or improper shutdown the flow cytometer will take several more minutes than usual to recover and return to the green light ready state This may occur on initial flow cytometer startup after installation It will also occur after an interruption of power to the unit 2 2 BD CSampler Software Workspace The main BD CSampler Software window is called the BD CSampler workspace The workspace contains controls and displays that provide access to all functions required for data acquisition and analysis The workspace is organized on five separate tabs Manual Collect Contains controls for setting up data collection and acquiring data in any order see chapter 4 for details Auto Collect Contains controls for automatically collecting data from several wells in order horizontally or vertically starting at a designated well see chapter 5 for details Analyze Allows analysis of multiple samples simultaneously See chapter 6 for details Statistics Displays statistical information see chapter 7 for details Batch Analysis Contains controls for analyzing batches of sample data see APPENDIX F for details 7820099 01 Rev 0 BD CSampler Software User Guide 2 3 2 4 BD CSampler Manual db E Plot 1 404 8pk verification run X a Plot 2 A04 Spk verification run Plate Name c a GATE No Gating GATE No Gating OE ERE At a2 na aa as as VA AB AB
137. re size e Click on the Set Core Size button in the Fluidics section of the Manual Collect tab e Move the slider to adjust the core size Core Size 3 Hm OK Cancel Figure B 2 Set Core Size 7820099 01 Rev 0 105 BD CSampler Software User Guide NOTE Certain core sizes are not compatible with certain flow rates BD CSampler Software does not allow these combinations to be set Use the following table to determine allowable combinations Table B 1 Core Size and Flow Rate Guide e Click on the OK button to set the core size and close the slider 106 7820099 01 Rev 0 BD CSampler Software User Guide APPENDIX C TRACKING USER ACTIVITY User tracking allows laboratory administrators to track the activities of flow cytometer operators by assigning a user name and password to each individual Passwords are created and used in BD CSampler Software and are unrelated to any Windows passwords used on the host computer or network User Tracking is an optional upgrade for BD CSampler Software and requires the use of BD CSampler Software version 227 or above C 1 Installing the User Tracking Module Ensure BD CSampler Software version 227 or above is loaded on the computer Copy the User Tracking Activation Key from the installation CD to the computer desktop Double click on the User Tracking Installer icon on the desktop ea Ami pA stellen ee Figure C 1 User Tracking Installer Icon In the installation
138. rounded to the nearest second during data export The Width parameter is that for the primary threshold parameter Table H 1 Example csv File 1 D Oo s om an 136 A FSC A 747359 308831 656756 519654 818135 257396 268765 741749 74788 310347 676065 611517 321267 301525 787700 712542 940993 93345 1120179 735806 93556 539263 390039 656006 749099 249477 B 55C A 206049 36583 159245 138403 257287 19821 16276 183316 19503 28687 201903 252169 42064 56315 328238 188615 240525 18538 898131 205623 28907 150426 401617 159032 186852 24622 FL1 A 785 390 746 767 203 168 329 295 405 585 788 149 314 926 567 871 65 6328 342 425 657 720 570 526 598 D FL2 A 147 221 179 329 72 254 145 204 213 500 242 194 133 92 224 110 1672 158 178 220 240 198 177 267 E FL3 A 0 456 870 225 1068 75 517 70 513 1374 244 442 1402 376 122 786 1878 776 1106 626 1270 433 437 757 F FL4 A 423 0 286 763 691 76 26 526 194 206 715 0 660 118 15 0 0 378 3020 565 217 435 542 0 374 0 G FSC H 1883688 787489 1935215 1175444 2171916 688493 742169 2209348 279103 811460 1755862 1461590 823497 734258 2011475 1939139 2740219 340737 3199136 2127351 312696 1257974 1519975 1809412 2095595 640125 H 55C H 704036 127133 555473 463213 8 6805 71597 58897 664629 76864 101016 709407 8897
139. s to ensure delivery of all components BD CSampler BD CSampler Mat BD CSampler SIP Collar Installation Removal Tool Mounting Bolts Figure 1 1 BD CSampler Accessory Kit Table 1 1 BD CSampler Shipping Contents BD CSampler BD CSampler Mat Mounting Bolts Installation and Removal Tool BD CSampler Collar 24 Tube Rack not shown BD CSampler Software not shown 7820099 01 Rev 0 1 BD CSampler Software User Guide To install the BD CSampler Turn off the flow cytometer CAUTION Failure to shut down the flow cytometer during BD CSampler installation could result in damage to both the flow cytometer and BD CSampler electronic modules Unscrew the SIP collar remove the sample stage and install the BD CSampler SIP collar Figure 1 2 Installing the BD CSampler SIP Collar Open the lid of the flow cytometer and locate the bolt holes for mounting the BD CSampler Bolt holes Figure 1 3 Location of Mounting Bolt Holes 7820099 01 Rev 0 BD CSampler Software User Guide e Hold the BD CSampler chassis with both hands and place the tab into the slot on the back of the flow cytometer Figure 1 4 Insert BD CSampler Tab into Flow Cytometer e Align the front of the BD CSampler so that the holes on the module are lined up with the three threaded holes in the flow cytometer e Hold the module with one hand and partially screw in the right mounting bolt to secure the BD CSampler to the flow cyt
140. sers delete existing users and change passwords C 3 1 Adding User Accounts 108 Sign in as the administrator Select About gt Users in BD CSampler Software NOTE The Users menu option is only visible to the administrator In the Users dialog box click on Add New User Users Username Password Notes Add New User Save Cancel Figure C 4 The Users Information Box Type the Username Password and Notes optional in the blank text boxes Notes are only visible in the Users dialog box 7820099 01 Rev 0 BD CSampler Software User Guide Users Username Password Notes admin Admin Lisa ann Arbor23 Department of Biomedical Sciences Delete Add New User Save Cancel Figure C 5 Adding a New User e Click on the Save button to save changes C 3 2 Deleting User Accounts e Select About gt Users in BD CSampler Software e Click on Delete next to the user account to be removed NOTE The administrator s account cannot be deleted Users Username Password Notes admir Admin Lisa Ann Arbor23 Department of Biomedical Sciences Delete Add New User Save Cancel Figure C 6 Deleting a User e Click on the Save button to save changes C 3 3 Changing a Password The administrator can change a user s password at any time To change a user password e Select About gt Users in BD CSampler Software e Delete the text in the Password field and type a new password e Click on the Save button C
141. seseeeeseeetaneenaees 87 Table 7 Lolglisiics Tab GOnWO S savannen 88 Figure 7 2 Creating Master Statistics Table Adding Plots cccccceecesseeeesseeeseeeeeseeeesseeeees 88 Figure 7 3 Creating Master Statistics Table Adding Samples cccccsececseeeceeeeseeeseeeeneees 88 Figure 7 4 Display Plot Preview LIS swsstivearscuevesaveeiesarasaiseasentistaaadllasassdiitaandlslacassdiitaaadlclasadones 89 Figure 7 5 Plot Preview uusesnsasaak sanne chactenccanceuxe vane deme cancsexe shan tencummoeencseantenc nes 89 Figure 8 1 The Run Backflush Cycle WiIndOW ccccccccseececeeeeceeeeeceecesaeeeeseeeesaaeeeseeeessaeeeas 91 Figure 8 2 The Run Unclog Cycle WInNdOW ccccccsecceseeeeceeeesseeeeseeeesaeeeeseeesaeeeeseeessaeeees 92 Figure 9 1 Before and After Applying VirtualGain rrronrrnrnnnnnnnnenrnnnnrnnnennnnrnrannnnnnnennnnnnnnnnen 96 Figure 9 2 Setting Up Histograms for VirtualGain rerrnrrvvrnnvrvrrnnrrernnrrvnrnnerennnrreernnerenrnnerennne 97 Figure 93 MONG PIOUS see 98 Figure 94 Pick th Sample IG Aligi vr 98 Figure 9 5 Move the Peak Definition Marker rrrnnrrnnnrnnnnennnnnnnnennnnennnnnnnnennnnennnennnnennunennssnnnne 99 Figure 9 6 Black Asterisk Identifier rarrnnnrrnnnnrnnnrrrnnennnnnvanrrnanrnnnrnnanennnnnnnsrnnnnennnsnnanennnsennn 100 Figure 9 7 Plot with Black Asle kv ee 100 Figure 9 8 VirtualGain Applied in an Overlay HistOQram ccccccsecccseecesee
142. stics and analyze results BD CSampler Software provides the following features Tabbed views for collection analysis and statistics Digital signal processing and color compensation at any time Drag and drop plots File export in FCS 3 0 format Seamless data file importation into FCS Express Batch Analysis of sample data Enhanced Analysis upgrade adds Drag and drop of publication quality images Event coloring Live gating 2 1 Starting BD CSampler Software Do not use BD CSampler Software until the BD CSampler has been completely set up see chapter 1 Introduction to BD CSampler To open BD CSampler Software e Double click on the BD CSampler Software icon on the computer desktop BD CSampler Software opens a new blank workspace e Select a plate type from the drop down menu 6 7820099 01 Rev 0 BD CSampler Software User Guide BD CSampler Manual Collect Plate Type v E Plot 1 No Sample 2 GATE No Gating Plate Name A01 c lick Here To Rename Sample E2 3 4 S 6 7 e 9 10 ED 0 200 000 400 000 600 000 800 000 Select a Plate Type gt T T T 500 000 1 000 000 1 600 000 FSC A Eg SYKE Lam mm og e ee 2 2 2 2 2 2 2 2 r 100 00 100 00 Cumulative r Events 0000 All 9 outside 1 1 z Data Capacity Used 0 of 96 000 000 Events Figure 2 1 New BD CSampler Software Workspace NOTE If BD CSampler Softwa
143. t 0 1 The specified wash cycles are performed after each sample acquisition To wash the SIP e Select the number of wash cycles from the drop down Wash Settings list Wash Settings None v Hone 1 Cycle aa Cycles 3 Cycles Figure 5 8 Wash Settings Controls e Click on the Apply Settings button NOTE The Apply Settings button also applies run limits thresholds and fluidics Wash settings can be applied to individual wells 5 6 6 Changing Run Direction Data can be collected from plates either horizontally A1 A2 A3 etc or vertically A1 B1 C1 etc To set the run direction e Doone of the following 7820099 01 Rev 0 73 BD CSampler Software User Guide 5 7 74 e Click on the Run Horizontally radio button in the Control Panel to collect the data horizontally e Click on the Run Vertically radio button to collect the data vertically Agitate Plate f Run Horizontally e None h after Whe ts C Run Vertically every hlin Figure 5 9 Run Direction Controls Creating the Sample Annotation Table The Sample Annotation Table allows users to name samples rename parameters and add a notation to each sample Information can be entered manually for each sample or copied and pasted from a spreadsheet program Sample Name Rename FL1 Rename FL2 Rename FL3 Rename FL4 Figure 5 10 Sample Annotation Table To manually create the Sample Annotation Table e For each sa
144. threshold values NOTE Run Limit settings are not carried over into the Auto Collect tab Collect data from a plate if the plate has been interrupted or aborted in the Auto Collect tab View sample data in plots and in the Statistics Table Print plots and statistics Perform color compensation Import and export data 4 1 Viewing the Manual Collect Tab The Manual Collect tab is organized into two major sections Instrument Control Panel Panel on the left side of the window that contains controls for collecting data Data display Large area on the right side of the window that shows the sample data in plots and in a Statistics Table 22 7820099 01 Rev 0 BD CSampler Software User Guide BD CSampler Manual Collect Plate Type 96 deep well plate ym te Plate Name c 3 A03 HPB CD3 CD45 SuSE NBG OEE 2 at a aa aa as AB AT AB AQ A10 A11 A12 B1 B2 B3 B4 BS BG BF BS BY B10 B11 B12 Plot 1 A03 HPB CD3 CD45 Plot 2 A03 HPB CD3 CD45 Plot 3 A03 HPB CD3 CD45 GATE No Gating GATE No Gating p GATE No Gating 10 000 12 800 SSC A Count 6 000 C1 C2 C3 C4 C5 C6 CF C8 C9 C101C110C12 2a t a g a D 2 a a 2 Ss a a a 2 a a a Ss a D3 D4 DS D6 D7 D8 DY D10D11D12 0 E1 E2 E3 E4 E5 E6 EF E8 E9 E10 E11 E12 i a Q 0 500 000 1 000 000 1 600 000 w ot gag 2 0 000 000 10 000 000 16 777 215 FSC A FSC A F1 rol ral ral rsi F6 F7 FS F F10 F11 F12 C
145. ting Master Statistics Table Adding Plots In the Sample Selector list select samples by enabling check boxes BD CSampler Software automatically adds the rows of samples to the Master Statistics Table and displays the sample data Sample Selector Master Statistics Table Addrows te your master ait Select cells from the Sample Selector and Statistics Column Selector to build your table table by selecting samples Saaz Sample Name Plot 1 FSC A SSC A Plot 2 FL1 A Plot 3 FSC A All Samples Events uL ful Mean F5C A FSC A 402 HPB CD45 PE Cy 7 420 020 6 cease 231 332 co 65 9 124 2 231 332 co 135 2 231 332 co 413 478 5 229 303 col 71 6 164 3 229 303 o 139 4 229 303 416 636 3 403 HPB CD3 CD45 404 HPB CD3 CD4 Figure 7 3 Creating Master Statistics Table Adding Samples 7820099 01 Rev 0 BD CSampler Software User Guide 7 3 Previewing a Plot in the Statistics Tab To preview a plot in the Statistics tab e Inthe Display Plot Preview list click on the appropriate plot to preview Display Plot Preview Plot 1 FSC AISSC A Plot 2 FL1 A Plot 3 FSC A Figure 7 4 Display Plot Preview List e Inthe Sample Selector list select the radio button of a sample BD CSampler Software displays the sample data in the plot Plot 1 402 HFB CO Gate Mo Gating 200 000 400 000 600 000 800 000 S00 O00 1 000 000 1 600
146. to Plot 1 FSC HISSC 4 a ny Plot 4 FLH review Sample Hame Noras RI M3 M4 M2 MS _ M7 At Samples Mean FSC H Mean SSC H CVFSC H MeanFLi H MeanFLI H CVFLI H Median FLI H Mean FLI H MeanFL2 H MeanFL2 H MeanFL2H CVFL C 7 AD Spk initial AO3Bpkafterunty 562 963 55 414 414 02 1 81 2 402 50 301 826 36 1 49 301 893 0 177 99 131 76 1 669 17 238 580 55 1 C r ag2sscunty ADA Spk verification run 562 708 27 414 372 89 1 80 240210 301 451 04 1 50 a 301 537 0 178 37 129 87 1 692 55 238 230 54 1 803 6pk after unity 580 276 78 521 627 05 1 44 2 170 24 301 904 00 1 32 303 017 o 234 05 159 16 1 224 49 238 291 93 1 C M AS Spk after unity ppsGpkevenfieationrun 579 439 63 522 022 34 1 50 2 173 81 300 479 72 17 23 301 429 5 236 95 158 86 1 232 95 238 203 17 i 7 ADA Spk verification BOS 578 896 91 522 310 46 1 53 2 188 34 304 304 68 142 304 638 5 237 88 159 26 1 240 80 237 037 17 i C B01 Gpe ntial C BO2FL4 Unity C V 803 6pe after unity C V 804 Sp verification C W 605 Figure 7 1 Statistics Tab Workspace BD CSampler Software User Guide 7 2 Statistics by plot Statistics by sample Click to add samples 88 The following table describes each of the controls and indicators in the Statistics tab Table 7 1 Statistics Tab Controls Previews the selected plot See section 7 3 for details Display Plot Preview List of plots imported from the Manu
147. tton is clicked and continues counting for 19 days 16 million tenths of a second even if data is added to the sample at a later time The time parameter cannot be reset to zero even by deleting data Agitating Samples The agitate feature is designed to maintain a sample in suspension not to resuspend a completely settled sample CAUTION Sample volume should not exceed 50 well capacity to effectively maintain suspension while avoiding spillover during agitation To perform an agitate cycle in the Manual Collect tab e Click on the Agitate button Copying and Pasting Plots To copy and paste plots from the Collect or Analyze tab to a Microsoft Office compatible application e Click anywhere on a plot and drag it to an open Microsoft application NOTE Ctrl C and Ctrl V cannot be used to copy and paste plots from BD CSampler Software into other applications If the Enhanced Analysis activation key is installed one of two file formats can be selected for plots during drag and drop actions To select the file format to use e Select File gt Set Plot Drag and Drop Format e Inthe dialog box choose one of the following formats e png when lower resolution is sufficient Drag and drop to Excel requires png format e eps when higher resolution images are required for publication or posters 7820099 01 Rev 0 BD CSampler Software User Guide Set Plot Drag and Drop Format Set Drag and Drop Format as
148. tware Template A BD CSampler Software template contains a predefined BD CSampler Software workspace for quick and easy setup and analysis All markers regions gates parameter names and sample names are saved without any data points BD Accuri provides several templates see the BD CSampler Software Installation CD thumb drive or the BD Accuri website at www accuricytometers com technical_information templates For details on creating custom templates see section 4 13 78 7820099 01 Rev 0 BD CSampler Software User Guide 5 12 Exporting and Importing Files Data from individual sample wells can be exported from any tab as FCS 3 0 files For details on exporting and importing files see section 4 19 5 13 Exporting Sample Settings Sample settings can be exported as a csv file for viewing data in a spreadsheet Sample settings include acquisition criteria sample names parameter names and compensation values To export sample settings e Select File gt Export Sample Settings a Save i Save In r BD Accuri CSampler v fr fmi 182 o Sampler 24 tube rack Template File Name Sampler 24 tube rack Template Files of Type Template c6t Figure 5 15 Export Sample Settings e Navigate to the location to save the file e Type the name of the file in the File Name field and click on the Save button 7820099 01 Rev 0 79 BD CSampler Software User
149. uadrant in the Statistics Table shown below Plot 3 Sample A Count Median FL1 A Median CD45 PE CyT A ae Gated on P1 in ally This Plot 101 660 al NN 96 737 4 841 0 45 399 0 Oo aR oa oo forsee 200 io Figure 4 47 Statistics Table Displaying Median Values Compare the median values of the affected channel If the median value of the UL or LR quadrant is not equal to the median value of the negative population LL fluorescence compensation should be applied 7820099 01 Rev 0 BD CSampler Software User Guide e Click onthe Set Color Compensation button in the Manual Collect or Analyze tab to open the Compensation Settings dialog box The dialog box contains four rows of FL buttons one row for each fluorescence channel Compensation Settings for HPB Auto CD11b CD45 Correct FL1 by subtracting a percentage of Le o oo Yo PS Yo Correct FLZ by subtracting a percentage of FL1 I Yo Flis I Yo Correct FL3 by subtracting a percentage of FL1 I Yo flees Yo Correct FL4 by subtracting a percentage of FLi Yo PL2 Yo Apply to Reset all f HFE Auto Apply amp Cancel amp ko 0 00 Alde Close Close f All samples Figure 4 48 Compensation Settings Dialog Box e Inthe row associated with the channel to correct click on the FL button of the fluorescence channel that is spilling over e f needed click on the Reset all to 0 00 button to clear all compensation values
150. ual Collect tab Plate Type Drop down list for setting plate type down list for setting plate type Load Plate Eject Plate Moves the sample plate into position to be loaded onto or ejected from the flow cytometer Plate Name Text box for naming the plate Sample Naming Field Text box for naming the sample Sample Grid Matrix laid out in the configuration of a 96 or 48 well plate or a 24 tube rack to correspond to sample vessel BD CSampler Software acquires each sample into its own well in the Sample Grid The wells can be filled with data in any order The wells are color coded e White Does not contain data e Blue Contains data Red outline Currently selected for viewing or collecting data 7820099 01 Rev 0 23 BD CSampler Software User Guide Traffic Light and Message Indicator that displays BD CSampler Software s readiness and system messages Before data collection can begin the software must display a green Traffic Light with the message C6 is connected and ready The Traffic Light status is color coded e Green BD CSampler Software is ready to collect data or is collecting data Yellow The flow cytometer is preparing to perform an action or a non critical error has occurred Red A critical error has occurred Run Settings Contains a set of controls that allow criteria definition for MN automatically stopping data collection See section 4 2 6 for details size See section 4 2 1 for d
151. ugh the plate or tube rack BD CSampler Software displays a small blue square in the upper left corner of each completed well A red border around a well indicates the sample that is currently being acquired The current well also flashes blue during data acquisition CAUTION Data is collected into all wells with settings applied even if they already contain data 7820099 01 Rev 0 75 BD CSampler Software User Guide 76 To begin data acquisition e Click on the AUTORUN button The status banner displays the current well and the message Events are being recorded The Run Display also updates the acquisition counters and the sample data in the plots BD CSampler Auto Collect a Gee Mo Gating A Count tao Figure 5 12 The Run Display While Collecting Data When data collection is complete BD CSampler Software displays DONE prominently across the Run Display BD CSampler Auto Collect Set The eshokt Delete events sa Figure 5 13 The BD CSampler Software Display After a Sample Run When a plate is complete click on the CLOSE RUN DISPLAY button to close the Run Display and continue using BD CSampler Software 5 8 2 Stopping Data Collection Data acquisition can be stopped by interrupting the plate or aborting the well 7820099 01 Rev 0 BD CSampler Software User Guide e Interrupt Plate Completes data collection from the current well and performs wash and agitate cy
152. verlays can be dragged and dropped into most Microsoft Office compatible programs Plot List Lists the plots that are available in the Analyze tab Available plots include plots copied from the Manual Collect tab or created in the Analyze tab Set Color Compensation Opens the Compensation Settings dialog box for correcting fluorescence spillover See section 4 14 for details 7820099 01 Rev 0 81 BD CSampler Software User Guide Plot Corrals Area displaying two rows of plot corrals Scroll up or down to view more plots For information on creating plots see section 6 2 2 Statistics Table Table below the plots that displays statistical information on individual plots Statistics Tables can be copied into most Microsoft Office compatible programs 6 2 Setting up Plots In the Analyze workspace plots can be created or copied from the Manual Collect tab Plots that are copied from the Manual Collect tab are appended with a C for example Plot 1C NOTE See APPENDIX F 4 for details on saving plots as publication quality images 6 2 1 Copying Plots from the Manual Collect Tab e Inthe Analyze tab click on the Copy Plots from Collect button e Inthe Copy Plots from Collect dialog box do one of the following e Select plots to copy by checking the box es e Enable the All Plots check box to copy all plots from the Manual Collect tab COPY PLOTS FROM COLLECT Plot 1 FSC A SSC A becomes Plot 1C Plot 2 FL1 A
153. ycle purges the cytometer with fresh sheath fluid and performs a backflush This cycle takes about five minutes To run a cleaning fluid cycle e Load a tube rack containing a tube of cleaning solution PN 653157 e Select Instrument gt Run cleaning fluid cycle 7820099 01 Rev 0 BD CSampler Software User Guide 8 4 8 5 Decontaminating the Fluidics BD CSampler Software automatically decontaminates the flow cytometer fluidics system at shut down The Decontamination Fluid Cycle can also be run manually at any time During this process the flow cytometer pulls decontamination fluid from the decontamination bottle then pulls sheath fluid from the sheath fluid bottle Decontamination takes about 13 minutes To manually decontaminate the fluidics e Load a tube rack containing a tube of water e Select Instrument gt Run decontamination fluid cycle Using the BD Accuri C6 Flow Cytometer for Precise Volume Measurements Each BD Accuri C6 flow cytometer is calibrated at the factory for accurate volume measurement before shipping Follow the guidelines below when accurate volume measurement is required during sample acquisition For more detailed information on this subject refer to the Technical Note A Guide to Absolute Cell Counting on the BD Accuri C6 Flow Cytometer available at www AccuriCytometers com e Ensure that the peristaltic pump tubing and in line sheath filter have been replaced within 60 days e Use 12x75 mm
154. yed 25013 125064 20 1 sid d 1 000 000 1 600 000 OO fe G Figure 4 17 Plot with Events Display Settings Applied 7820099 01 Rev 0 BD CSampler Software User Guide 4 7 Using Gates and Markers A gate is a specified area within a plot that is used to designate a set of events for analysis BD CSampler Software allows creation of any of the following types of gates Polygonal gate Gates an irregularly shaped area around a population of events Rectilinear gate Gates a rectilinear area around a population of events Quadrant gate Gates the plot in quadrants Vertical marker Gates a histogram plot to the right or left of a vertical marker Horizontal marker Gates a histogram plot within the upper and lower boundary of a horizontal marker 4 7 1 Creating a New Gate To create a gate in a density or dot plot Click on one of the following gating tools e Polygonal Gating Tool lt typically used for irregularly shaped populations e Rectilinear Gating Tool typically used for evenly shaped populations Quadrant Gating Tool typically used for analyzing fluorescence plots Use the mouse to draw a region labeled P1 for a polygonal gate R1 fora rectilinear gate or Q7 for a quadrant gate To draw a polygon click on the mouse to anchor each vertex and double click to close the polygon NOTE Gate labels can be changed by double clicking on the label and typing a new gate name in the di

BD CSampler™ Software User Guide

Contents

Download Pdf Manuals

Related Search

Related Contents

BD CSampler&trade; Software User Guide

Contents

Download Pdf Manuals

Related Search

Related Contents

BD CSampler™ Software User Guide