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CFlow User Guide

1. Ooo O J O O C Plot 1 FSCHISSC H i iQ 0 T Mm v Plot 1C 2 F5C H SSC H of This Plot of All Mean FSC H Mean SSC H Plat 2 FLI H pap E a a E a a E a a Plot 2C FL1 H Ce o 0 0o 0 E Plot 3 FL2 H Count of This Plot of All Mean FL4 H CY FL4 H Plot 3C FL2 H TthisPlot CE O O S O y O O C Plot 4 FL3 H Pome S O S O S O O C Plot 4C FL3 H Om o o ooo S O S O y O O E Plot 5 F5C H 55C H mM a a 7 Plot 5C FSC HISSC H Count CV Time Plot 6 FL4 H eS oo a C eye nt R fTimelFSC H Count nf This Plat a oF All Mean Time Mean FSC H Master Statistics Table Select cells from the Sample Selector and Statistics Column Selector to build your table Plot 1 FSC H SSC H Plot 2 FL1 H Plot 3 FL2 H Plot 4 FL3 H T FSC H CV h a MeanFSC H CVFSC H MeanFL1 as PH Mean FL3 H CV FL3 H increased from 2 to 092908 515 456 83 2 0 299 689 1 7 221 889 4 2 4 515 617 6 2 0 299 609 3 9 1 5 222 835 6 2 3 515 947 4 2 2 67 199 1 6 219 212 9 2 3 514 650 9 2 5 298 968 1 7 222 357 6 2 4 512 572 4 2 0 298 793 9 1 8 267 940 8 1 7 222 880 7 2 3 510 249 3 3 0 f 303 360 3 2 1 271 891 2 2 1 230 636 5 2 7 515 858 0 2 0 300 724 2 1 6 266 939 3 1 6 222 241 3 2 3
2. Set Color Compensation Gated on P2 in all This Plot 101 071 100 0 43 9 254 1 42 823 7 72 9 34 5 R2 87 747 86 8 38 1 278 7 44 226 9 59 4 27 7 Q8 UL 96 806 0 0 95 8 42 1 252 6 44 669 4 70 2 27 on 0 0 0 0 0 0 0 0 0 0 0 0 0 O C Q8 LL 4 262 0 0 4 2 1 9 282 8 930 6 87 0 65 6 Q8 LR 3 0 0 0 0 0 0 7 414 0 1 883 7 26 6 55 5 Gated on R2 in P2 in all This Plot 87 747 0 0 100 0 38 1 278 7 342 2 59 4 93 2 N Q9 UL o 0 0 0 0 _ 0 0 0 0 0 0 0 0 0 0 a Figure 4 9 Analyze Tab Plots are selected for printing www AccuriCytometers com Page 40 Make an Overlay Histogram in Analyze Tab 1 Scroll down to an empty plot corral and select it background will turn from blue to white 2 Click on the Overlay Tool under the 96 well grid and a single parameter FSC A plot will open Figure 4 10 3 Click on the axis label FSC A and change to the desired parameter Use FL1 A if working with the HPB 4 Color Tutorial file Click on the Gate button and apply the desired gate R1 in P1 in all for the HPB 4 Color file Select the data wells from the 96 well grid to be overlaid Choose samples A2 A3 and A4 if using the HPB 4 Color file Figure 4 11 File Edit Display Instrument About Clow Plus Analyze Plot 8 No Sample GATE No Gating n 1 2 3 4 5 6 7 8 9 10 11 12 Default Overlay M A2 AB AA 45 AB A7 AB AD AIO A11 A12 Plot B1 B
3. Coo io 517 1916 3 9 294 099 4 2 4 261 867 6 2 4 219 198 9 2 9 Figure 1 8 Statistics tab Display of 8 peak bead data from successive days Troubleshooting If there are very broad or multiple peaks for FSC H fewer than 8 peaks visible on FL1 H and or FL2 H fewer than 6 peaks on FL3 H or the brightest peak CVs are gt 5 0 in any channel check the following 1 If bead data was acquired using the Slow rate Under Fluidics Control in the Control Panel If not select Slow re suspend the beads by vortexing or flicking the tube and recollect the data 2 The age of the beads If the beads have been diluted for more than 2 weeks kept at room temperature or warmer or exposed to light for long periods of time their performance may be degraded Make up new bead suspensions and run the bead sample again 3 If there is a bubble or clog in the flow cell a Run the bead sample again b Remove the tube from the SIP place an empty tube under the SIP and click Unclog When the Unclog cycle is finished Traffic Light will be green run the bead sample again c Remove the tube from the SIP place an empty tube under the SIP and click Backflush When the cycle is finished Traffic Light will be green run the bead sample again For further troubleshooting refer to FAQs at www AccuriCytometers com or call Accuri Technical Support at 1 734 994 8000 www AccuriCytometers com Page 12 Chapter 2 Getting the Mos
4. ccceecccccecceeeeseeeeeeeeceeeeeeeeeeeeeeeseeeseeeeeesseaeseeeeeeesssaeegeses 52 Figure 6 6 Step 11 Move the Peak Definition Marker ccccccccccceecceeeeeceeeeeeeeeeeeeeeeeeeesseaeseeeeeeessaaaaneees 52 Figure 6 7 Step 14 To align other samples exactly as the first aligned sample ccseeeeeeeeeeeeeeeeeees 53 Figure 6 8 Step 15 A black asterisk appears under the Sample to Align plot ccccccssseesseeeeeeeeeeeeees 53 Figure 6 9 The sample with VirtualGain applied has a black asterisk under the parameter label 54 Figure 6 10 Click the asterisk to switch between VirtualGain and native displays A grey asterisk indicated native data a black one Virtual Gain ADPlied cece ccccccececeseeeceeeeeeceaeeeeeeeeeeeesseeesseeeeeeseueeseeeeeeeesssaaaaeees 54 Figure 6 11 Overlays automatically display VirtualGain whenever applied ccccceeeceseeeeeeeeeeeeeeeens 55 Figure 7 2 The Plate Type drop down MeN sceeeceecceeeeeeeeeeaeeeeeeceaeeeceeeeseaeeceeeeseaeeeeesseeeeeeeseaeeeseeeeaas 57 Figure 7 1 CFlowSampler start up SCreen cccccsseeseecceceeceseeeeeeceeeeeseaaeeeeeeeeseseaaeseeeeeeessaaaaeeeeeesessaaaaesss 57 Figure 7 3 The CFlowSampler Manual Collect Tab ccccccccccccccccceeceeeeeeceeeeeeseeeeseeeeeeessaeeeseeeeesssaeeeeeees 58 Figure 7 4 The Run Backflush Cycle Window ccccccccccecceeeeseeeceeeeeeeeeseeeeeeeessueeseseeeeeeesseeaseeeeseeesaana
5. 610 3 139 4 30 8 c C A Mean CD45 PE Cy7 A CVC lot 10 796 4 41 303 7 UL 2orsTo To ToT TOT 59 3 33 591 3 JR 75 980 0 0 75 7 32 8 28 595 4 45 234 2 Figure 3 33 Gating Plot 2 CD3 FITC vs FL2 A to include events in gates R1 and P1 www AccuriCytometers com Page 33 CFlow Plus accuri A es Plot 2 Sample HPB CD3 GATE R1 in P41 in al A4 HPB CD3 F CD4 CD45 CD8 Plot 1 Sample HPB CD3 Plot 3 Sample HPB CD3 GATE No Gating GATE P1 in all N C I 2 3 4 5 6 7 B 9 20 11 12 E A m AB AB AT AB AQ A10 A11 A12 EEES i k B B1 B2 B3 B4 BS B BF BS BY B10 B11 B12 xi C C1 C2 C3 c4 i c5 i cog c7 csi co c10 c119Cc12 q Ir aA D D1 D2 D3 D4 DS DEG DF DB DE D10 B11 B12 7 a t a E E1 E2 E3 E4 E5 EG EF E8 E9 E10 E11 E12 PNE g a F F4 F2 F3 F4 F5 FG FF FS FO F410 F114 F12 4 Ss G 61 62 63 64 G5 GH 67 GB 69 G10 611 612 3 wt dwt ad H H1 H2 H3 H4 H amp S HG H7 H8 HY H10 H11 H12 FSC A CD3 FITC A 1 te 1e C6 and CFlow are connected BPEC ON Aue e and ready C Plot 4 Sample HPB CD3 Plot 5 Sample HPB CD3 Plot 6 Sample HPB CD3 Run Limits a GATE P1 in all al GATE P1 in all l GATE R1 in P1 in al m Slow Medium Fast Flow Rate 14 pL min kE Ei 3 v 125000 events Core Size 10 pm Custom at Pe ES in Ungatea Sample
6. P virtual Gain Tutorial CFlow File Edit Display Instrument About Cflow Plus Plot 10 Sample 41 Plot 11 Sample 43 GATE P2 Plot 12 Sample 41 03 Af 82 8 ma 95 oa AF HA 99 AO M M12 BI 62 B0 Ba 6S Ba B7 63 B9 BIO B1 612 c1 e2 ea ca les lce c7 ca c9 cicne D1 D2 D3 Da DS D6 D7 Da D09 D1001 D12 10 000 Ei E EJ Ea E5 E EF E E9 E10 E11 E12 Fi F2 FQ Fa FS FG F Fa FO FIO F11 F12 a2 A amp SG GC G8 9 GINGIIGI2 Hi H2 HJ Ha HS HG H7 HA H9 H10H11 H12 Make anew plot Aj fal COPY PLOTS FROM COLLECT Plot List Plot 5C FL1 A 5 000 Count F5C A S5C A FLI A FL2 A FL3 A FL4 A Plot 12 FL1 A Height gt Selecta iah Select a plot and Select a plot sample t _ Time sample to analyze sample to an lt Rename Parameters V irtualGain Figure 6 3 Click on the histogram parameter label to reveal the Parameter list www AccuriCytometers com Page 50 6 Select VirtualGain from the Parameter List The VirtualGain dialogue box will open Figure 6 4 7 Move the Peak Definition Marker light blue vertical line in the Standard Sample plot to the center of the peak that will be the reference point Other sample s will be aligned to this position The plots in the VirtualGain dialogue box will be Zoomed to the same axes ranges as they are displayed in the Analyze tab To Zoom in further or scale over a different range use the Zoom Tools in the Anal
7. HPB Auto CD45 A1 A2 plots c Documents E HPB Auto CD45 A1 A2 c6 E HPB CD3 F CD4 CD45 CD8 c6 HPB CD3 F CD45 PE Cy A04 c6 Save as Template E HPB Unstained At c Import FES File 4i H Human PB c6 Export Fics File 7 6 g Export 4LL Samples as FCS AT AB AD Print Selected Items Ie B7 BS B9 Quit CF CS Cg Fil i uman PB 5 ul Pc ile name PB save m P Pe Places Files of type cFlow c6 Cancel E E1 Ez E2 E49 ESF EG E7 f ES f ES a Naming the CFlow File b Choosing Save CFlow File Figure 3 19 Saving and naming a CFlow File Saving data as a CFlow file will save the CFlow workspace in its entirety including sample data collected along with plots gating compensation and threshold settings Not only will the Collect tab settings be saved but so will any changes made in the Analysis or Statistics tabs A CFlow file is a comprehensive and often large data file that saves instrument settings FCS files and plot layouts together Data collected on the C6 should always be saved as a CFlow file CAUTION CFlow does not automatically save CFlow files to the computer s hard drive The data is only saved locally in RAM until the user saves the data as a CFlow file 2 After the CFlow file name is applied it will appear in the upper left corner of the CFlow file workspace Figure 3 20 www AccuriCytometers com Page 26 mr CFlow File Name gt File
8. Introduction to CFlow and Running the C6 The C6 Flow Cytometer system includes CFlow or CFlow Plus software for controlling the instrument acquiring data generating statistics and analyzing results Throughout this User Guide CFlow refers to both CFlow and CFlow Plus unless indicated otherwise CFlow Workspace The CFlow workspace provides access to all the functions needed to acquire and analyze data CFlow is divided into three separate tabs Collect Analyze and Statistics This section focuses on data acquisition and the Collect tab The Collect tab appears when CFlow is launched and is used to acquire data from samples processed on the C6 The Collect tab enables physical control of the C6 provides access to settings needed to specify the threshold and stop criteria as well as many analytic functions Figure 1 1 shows a partial Collect tab The Sample Grid is laid out in the configuration of a 96 well plate to facilitate both organization of experiments and data acquisition from sample tubes CFlow acquires each sample into its own well in the Sample Grid The wells can be filled with data in any order that is convenient White wells do not contain data and are available to use for data collection Blue wells contain data A red outline around a well indicates that well is currently selected for viewing or collecting data Instrument Controls The left hand column of the CFlow Collect tab provides access to all instrument controls ne
9. of fo i E HF HA HE HD HF HE HE HBOT HIN HET Flik Gi snd fiya ma corme ed Flat E lopie liner GAE M im sij Pora impii inenika GATE M in wt Fluidics fiw O Wiium C fen Fima Eite t plisin Coie Tie 10 pm fot et te oe FLEA ao oe Pid Semple Umetained Coumi Voluma pl Sof Thi Piot Sonal M n SCA Mean SSCA GWTSCA CYSSC A Madunist a AM 123 064 EE ee en Far enn MA Fl 7 al EE Ie Ia ADI i et HAL I PA 2 Sample Ueetained Court Volume ul ol This Plot Sofa MeanFLLA MeanFL7 A CVPLIA CVFLAA Medan FLtA Met a Evens LIHA 7 noe Tew ppg F Wais bater dating Gated on Pl in ay Ta pam a This Plot 47 7208 a ama T H T IEE au Saa HA G Cwertes Se 6 Cane Capacity lad MA i l 1 1 ef 90 000 Evan en O60 iers iyl ia 70 000 000 Ever l Mei E Sampl liesiained Coumi Volume pl Sof Thi Pot Sof Al MeanFli A Mean FLIA CW RLA CV FLIA Median FLiA Mer Gated on FH in a This Piee 47 238 0 0 100 0 37 344 7 BAT ai an MA Phot Semple Uneteined Court Volume pl Sof This Pict Sof All Mean FLIA Mean FLUKA CWFLIA CVFLEA Median FL2 A Mex Gaurd en Fi in a Tes FACE 47 ecw 0g 1T Fr NLG ee i fo 1 MHA a Figure 3 15 A CFlow Workspace with 5 added 2D fluorescence plots Plots required for tutorial FL1 A vs FL2 A FL1 A vs FL3 A FL2 A vs FL3 A FL4 A vs FL3 A FL1 A vs FL4 A Adding Data to a CFlow File and Naming Plot Axes 1 Colle
10. rlIOw CFlow User Guide Science is hard Flow cytometry should be easy Table of Contents Chapter 1 The Basics Operating the Accuri C6 Flow Cytometer c cccccssssesssesesesssereeiseserereeeen 4 Introduction to CFlow and Running the C6 cccceecceccceeceseeeeeeeeeeeeeeseaeeeseeeeeeeesseeaseeseeeeeessaaeeeeeeeeesaaas 4 LOW WOR AC eaea carbene E E EE EE E E A E 4 SN e E E E A E E E E E 4 RUNNING Validation Beads sirisser innii n a aana iaai a AREARE TEERAA ERR eeni AAE 6 SIVA Ee Dal Ta E EEE E EE E 7 Analyzing and Recording your Validation Bead Data ccccccccccccssseeeeeeeeeseeeeeaeeseeeesseeeseeeeeseaeeeeeeeeeess 8 FOUE SIC OTA se neces ce seston E E EE E A N 12 Chapter 2 Getting the Most from the C6 cc ccceccccccceeecceeeeeeeeeeeeeeseeeeeeeeeeessaeeeseeeeeeessaaaseeeeesessaaeaeeeeeeeees 13 Materials Needed to Operate and Maintain the C6 ccccccecececcceeeseeesseceeeeeesaeeeseeeeeeeessseeeeseeeeeeesaaas 13 System Cleaning and Validation 0 0 0 cece ccccccccseeeeeeeeeeeeeeeaeesseeeeeeeeeeeeeeeeeeeesseeeseeeeeeeeseasseeeeeseeessasaaaeees 13 Standard Operating Procedures for the CG ccccccccccecccssesseeceeeeeeaeeeeeceeeeessueesesceeeeeesseaaeeeeeeesssaaaaeesees 14 Routine Instrument Maintenance cccccsseceecceeeeeeceeeeeeeeeeeenseeeeeeseeeeeeeeeeeeseaeeeeseeeeeessaeeeesseseeeesseneeeeas 15 Tips on Data Collection and Analysis with the C6 ccccccccccccccssssece
11. 10 11 12 Es A J sh hi of f Alla a2 as aa as as ar a ag aoanan B1 B2 B3 B4 BS BB B7 Bs BO B10 B11 B12 a B 61 B2 B3 B4 B5 B6 B7 BS BS B10 B11 B12 A C1 c2 C3 c4 cs csl c7 csi cgl colc c12 a EE c1 f c2 f csi c4icsi ce f c7 i ce co iciaoicsssci2 A D1 D2 D3 D4 DS D6 DF DS D9 D10 D11 D12 A D 01 D2 D3 D4 DS DB D7 DS D9 D10 D11 D12 A E1 f E2 E3 E4 es f es EF E8 E9 E10 E11 E12 poe E E1 E2 E3 E4 E5 E6 EF ES E9 E10 E11 E12 pn 4 ral rol eo ra rsl rel rz rel ro lF F112 A F F14 F2 F3 F4 FS F6 F7 F8 FO F10 F11 F12 4 61 62 63 64 65 GB G7 68 69 61016111612 G Gi G2 63 64 G5 G6 G7 G8 GO G10 G11 G12 4 H1 H2 H3 H4 HS HG6 H 7 H8 HO H10 H11 H12 Lae H H1 H2 H3 H4 H5 H6 H7 HS HG H10 H11 H12 B B cB and CFlow are connected Z2 C6 and CFlow are connected m and ready B and ready B B B Run Limits Fluidics E Run Limits Fluidics B Run Unlimited Slow Medium Fast m Run Unlimited Slow Medium Fast BE B z i Flow Rate 14 pLimin m Flow Rate 14 pLimin fiooco Iv 10000 events ea ee 40 B Iv 10000 aani Core Size 10 pm B in unaated Sampie open ae in ungated Sample 1 Faa Teea Figure 7 9 Applying settings to a set of samples will turn those sample wells the same color Each new set of samples or sample settings will be displayed with a different color Figure 7 10 There are 12 colors available however up to 96 sets can be created The color
12. 3 De select the Include box next to the P1 on All Events option and select the Include box next to the option R1 in P1 in all Figure 3 33 www AccuriCytometers com Page 32 a OO SS Select the Apply button followed by the Close button at the bottom of the dialog box Repeat steps 1 to 4 above for Plot 6 CD3 FITC vs FL4 A Move to well A4 and collect the data for Sample 4 Save the changes to this CFlow file by selecting File Save CFlow File and overwriting the current file See Figure 3 34 for a view of the Collect tab after application of the light scatter and CD45 PE Cy7 positive gates to Plots 2 and 6 and collection of data for Sample 4 1 Click on Gate button 2 De select Include Option for P1 on all events 3 Select Include Option for R1 in P1 in all Select Apply Select Close Plot 2 Sample A4 CD3 F GATE R1in P1in al s3 s cozi FITC Change Gating for Plot EJ Gating choices 0 Include u w2 Pi FSC A SSC A P1 on all events FSC A SSC A R1 in P1 in all FL1 A FL3 A R1 on all events FL1 A FL3 A wt af w CD45 PE Cy7 A s Plot 3 Sample A4 CD3 F GATE P1 in all Plot 6 Sample A4 CD3 F CA 673 9 GATE P4 in all wo nt a8 CD3 FITC A aot iT 133 5 CV CD3 FITC 61 7 B48 1 2 265 9 33 2 B01 8 2 391 2 94 1 669 1 109 8 48 4
13. 3 dat u Q1 Compensation Settings for Sam g 00 Correct FL1 by subtracting a percentage of 250 iz 0 00 Fi ISS eo Fis 0 00 Correct FL2 by subtracting a percentage of afp Fis fpo Fis 0 00 Correct FL3 by subtracting a percentage of Speak ie 0 00 riz 0 00 Fls 000 wi a2 Correct FL4 by subtracting a percentage of Q an ale 000 FL2 0 00 Abs H 0 00 Apply to Sample 42 ati Save amp Cancel amp Close Close All samples E T 1 e pe 3 g o 2 a Median CD45 PE Cy7 A NA 42 578 83 34 0 3 96 751 0 0 95 2 42 0 2 054 5 44 694 0 26 6 45 395 0 1 0 0 0 0 0 0 12 774 0 62 966 0 0 0 62 966 0 4 908 0 0 4 8 2 1 183 3 877 5 70 9 69 2 767 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 E Figure 3 28 Results of selecting Plot 4 applying Quadrant markers and calculating Median fluorescence channel values www AccuriCytometers com Page 30 10 Choose the FL3 button in the second row of the Compensation Settings dialog box under the line Correct FL2 by subtracting a percentage of in order to input the appropriate subtraction value 11 Enter a subtraction percentage and click the Preview button in order to update the Statistics data For this example the appropriate subtraction percentage is reached when the Median FL2 value in Q1 UL is equal to that of FL2 in Q1 LL
14. CFlow Plus only Backflush and Unclog Controls Opens Color Compensation Matrix Real Time Acquisition Counter Display Collect B7 i2 3 4 5 6 7 8 9 109 11 B4 BS BG BS B9 B10 B11 C4 C5 Ch CF Ce C9 C10 C11 D4 DS DA D7 DS DS D10 011 E4 ES EG EF ES ES E10 E11 F4 Fo FG FF FR FO FIO F11 G4 65 Gh G7 GS Go 610611 L 7 mh AOA 0 p H4 HS HE HF He HE H10 H11 C6 and CFlow are connected m and ready Run Limits Fluidics Slow Medium Fast Flow Rate 14 pL min Core Size 10 pm Custom k 2 min 0 Sec ea Rae atin m 100 uL Core Size pm Backflush Unclag Threshold Set Threshold 20 000 on FSC H Hone in Ungated Sample Set Color Compensation Last Run Cumulatwe Delete Sample Data o o Events 0 00 Time 0 0 Microliters Oo 0 Events Sec Data Capacity Used 0 0 Events pl I 1 of 10 000 000 Events Il Warn before deleting Figure 1 1 The CFlow Control Panel in detail Sample Naming Field Fluidics Control Sample Rate Adjustment Custom Fluidics Settings User Defined Flow Rate and Core Size User Defined Stop Criteria CFlow Plus only Set Threshold Default is 80 000 on FSC H Run Click to Start Sample Acquisition Reads PAUSE during acquisition reads ADD TO when the well is done and or already contains data Delete Permanently Deletes all Events from Sample 4 Replace the tube on Sample Introduction Probe SIP with a fresh tube containing at
15. FL2 A includes events in gates R1 and P1 after following steps 1 3 in Adding and Applying Additional Gates Adjusting the Axes View Using the Plot Spec Tool Zooming When CFlow plots are viewed with logarithmic scaling data is displayed by default from channels 10 to 16 7 x10 For most analyses only a small section of the axis needs to be viewed In this section the Plot Spec Tool will be used to select the desired channel range 1 4 In order to better visualize the lymphocyte populations the axes range for both Plot 2 CD3 vs CD4 and Plot 6 CD3 vs CD8 should be adjusted Figure 3 35 blue boxes Plot 2 Sample HPB CD3 Plot 6 Sample HPB CD3 GATE R1in Ptinal i GATE R1 in P1 in al F z w a t a o wast ae wo asd CD3 FITC A CD3 FITC A oes A Go Ge wes A E Figure 3 35 Blue boxes show desired 4 log channel to view for Plots 2 and 6 Click on the Plot Spec Tool under Plot 2 Figure 3 36 Under X Axis set Min Value 100 and Max Value 1 000 000 Under Y Axis set Min Value 10 and Max Value 100 000 www AccuriCytometers com Page 34 5 Select the Apply button to view the changes and then the OK button to close the Plot Spec dialog box Plot 2 Sample HFE COz GATE FA in P1 in al Min value Max Value f log 100 1 000 000 Min and Max Channel Values fc linear Plot Spec Tool Min Value Max Value log Hide 1st 10 100 000 rj a
16. Run for 2 minutes in an empty data well to clear the last sample s residue from the SIP Leave the tube on the SIP after the run has finished Untitled Workspace CH File Edit Display Instrument Abo Open CFlow File or Template New CFlow File Save CFlow File Save as Template Import FCS File Export FCS File Export ALL Samples as FCS o Quit Figure 1 3 Saving a CFlow file Analyzing and Recording your Validation Bead Data Analyze the bead data using the Collect tab of CFlow 1 Click on the well in the Bead File which contains the 8 peak bead data to select it will be A1 if following previous procedure 2 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 clicking on the region it will have thicker lines when selected and dragging it so it looks like Plot 1 in Figure 1 4 This should contain 75 85 of all events in R1 There is usually a shadow population slightly higher in FSC H than the main cluster of beads this is normal for these particular beads bead doublets or clumps Do not include this group in R1 3 View the next 3 plots FL1 H FL2 H and FL3 H They should be gated on the scatter region R1 The phrase R1 in all should be visible next to the gray GATE button at the top of each of these 3 fluorescence plots Figure 1 5 If not click the GATE button and select the option R1 on all
17. Sample 6 Peak Be meee S GATE R1 in all GATE No Gatin o GATE R2in all Run Limits Fluidics i ee E Slow Medium Fast Flow Rate 14 pL min J 50000 events Core Size 10 pm Custom in Ungatea Sample M fo Min 0 Sec Flow Rate E fo O pL Backflush Unclog Threshold Set Threshold 80 000 on FSC H None Plot 5 Sample 6 Peak Beads 2 9 09 Volume pL 50 9 100 0 603 042 2 Set Color Compensation R2 50 9 85 5 588 005 9 Last Run Cumulative Delete Sample Data Plot 6 Sample 6 Peak Beads 2 9 09 Volume uL of This Plot Mean FL4H Median FL4H 0 Events 50 000 R 0 00 Ee 3 45 Wan before deleting Gated on R2 in all 0 0 Microliters 50 9 This Plot 100 0 85 5 209 149 2 0 Events Sec 222 Data Capacity Used M4 580 054 0 731 079 0 16 3 13 9 653 373 9 0 0 Events pL 982 1 1 of 10 000 000 Events LZIN Figure 1 7 Example of 6 peak SPHERO APC Calibration Particles shown in FL4 H The peaks in FL1 H FL2 H and FL3 H are not relevant Using the Statistics Tab in CFlow to Generate a Table of Mean Peak Channel and CV values A convenient way to monitor the Validation Bead data and thus the C6 performance is to store the 8 and 6 peak validation bead data in a single CFlow file saving the data from each day in its own well Then using the Statistics tab a table can be created of the Mean channel numbers and CVs for the top peaks a
18. or lost from the data file 3 The Traffic Light below the 96 well grid in the upper left hand corner should be green with the message C6 and CFlow are connected and ready When CFlow is opened sample well A1 will automatically be selected as indicated by a red border Name the current sample by typing into the text box above the 96 well grid Naming samples is optional and can be done at any time If no text is added the sample will be named according to the www AccuriCytometers com Page 18 well location e g Sample A1 Set a Run Limit For the first sample a good starting point is to collect 100 000 200 000 ungated total events The Run Limit can be changed once a gate has been set on the population of interest Additionally data acquisition can be stopped at any time or volume limit Multiple run limits can be chosen the C6 will stop on the first limit reached Set the Fluidics Rate to Slow The maximum data rate that the system can accommodate is 10 000 events per second To ensure the best data resolution it is recommended that samples be acquired at a rate of 5 000 events per second or less when possible Once the data rate has been determined the setting can be adjusted to Medium or Fast if necessary The default value for thresholds is 80 000 If it is not already set at this value set the Primary Threshold to channel 80 000 on FSC H Figure 3 3 Click on the Set Threshold button within the Fluidics control
19. 015 3 582 3710 2 1 6 5 vents 50 000 0 00 Time 3 26 V Warn before deleting 0 0 Microliters 46 6 z O Events Sec 242 Data Capacity Used Plot 2 Sample 8 Peak Beads 2 9 09 Count Volume uL of This Plot of All Mean FL1 H CV FL1 H Median FL1 H 0 0 Events pl 1 073 4 1 of 10 000 000 Events me This Plot 43 747 46 6 100 0 87 5 71 516 6 164 6 NA Mi 269 370 0 417 735 0 5 491 46 6 12 6 11 0 333 909 1 2 8 NA Plot 3 Sample 8 Peak Beads 2 9 09 Count Volume wL of This Plot of All MeanFL2 H CVFL2 H Median FL2 H Gated on R1 in all This Plot 43 747 46 6 100 0 87 5 60 081 4 172 2 NA M2 254 995 0 359 801 0 5 483 46 6 12 5 11 0 295 210 5 2 6 NA m Lo 2 LLLULLULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULULlCUDEdLeeae Figure 1 6 Example of 8 peak bead data collected with a C6 The number of expected peaks is FL1 H 8 FL2 H 8 and FL3 H 6 The peaks in FL4 H are not relevant Select the well containing the 6 peak bead data should be well E1 Adjust the pre drawn region R2 in the second FSC H vs SSC H to encompass the main data peak similar to the procedure for the 8 peak beads This population should look like an exclamation mark The R2 region should encompass both major populations Figure 1 7 8 Confirm that the FL4 H plot is gated on the region R2 If not click on the GATE button and select the option R2 on all events from
20. C12 D1 D2 BS D4 DS De D7 DE GS D10 D11 Biz E1 f E Es f E41 ES f EG f E7 f ES f ES E10 E11 E12 Fl F2 g FS Ft FS FR FR Fe FO FIO FAT Fiz G1 G2 Ga Gg 65 Gh GF GB G8 G10 6411 G12 L Tnm Oooo P Hi H2 H3 H4 HS He HF HB Ha H10 H11 H12 Figure 5 2 The Edit Menu Undo Undoes the last action that was performed in CFlow Shortcut Ctrl Z Not all actions are undoable For example deleting data using the Delete Sample Data button cannot be undone using this command Redo Redoes the last action done Shortcut Ctrl Y See Undo Copy Allows for a copy of a marker or region to be pasted into another plot It also allows for copying of statistics from the tables in CFlow to other programs such as Microsoft Office Shortcut Ctrl C To Copy and Paste plots from a CFlow workspace left click anywhere on a plot and drag it to an open application Ctrl C cannot be used in conjunction with Ctrl V to copy and paste plots from CFlow into other applications Paste Allows for the pasting of previously copied markers and regions into new plots Shortcut Ctrl V Ctrl V cannot be used in conjunction with Ctrl C to copy and paste plots into other applications Rename Parameters Allows renaming of individual parameters in either the current sample or all samples at the same time The dialogue box shown in Figure 5 3 will appear when this option is selected This option can also be accessed by clicking on a parameter label within a plot x Rename
21. CSampler will automatically move the arm and plate platform to Eject position After placing a container under the SIP to catch any fluids that will be ejected from the SIP click the Unclog button in the Run Unclog Cycle window Figure 7 5 Run Unclog Cycle Place appropriate container under SIP to catch Fluid Cancel Figure 7 5 The Run Unclog Cycle window Calibrate Fluidics The fluidics of the C6 can be calibrated for the volumes typically run from a 96 well plate This operation requires that at least 150 uL is placed within the well Once a plate is prepared with the appropriate volume load it onto the CSampler Upon selecting Calibrate Fluidics a window will indicate which well the C6 is going to use for the fluidics calibration Figure 7 6 This well will also be indicated by a red box in the Sample Grid If this is not the desired well switch to the Manual Collect tab and select the proper www AccuriCytometers com Page 59 well After the proper well is indicated click Calibrate The calibration will take several minutes Refer to the Accuri C6 Instrument Manual for detailed information Calibrate Fluidics alibrate Fluidics For 4 96 well plate plate type using currently selected well 41 Cancel Figure 7 6 The Calibrate Fluidics window Using the Manual Collect Tab The Manual Collect tab is designed for three purposes 1 To run samples individually from any plate type or tube rack When using this t
22. FLI to 0 L linone Rename Fl to FL linone Rename FLI to Oo A J inon Rename FL4 to O inon l Apply to f Hao Sample OK Cancel i All samples Figure 5 3 Rename Parameters Once a parameter has been renamed that name will appear as an option in the drop down menu under the word Select right hand column of the Rename Parameters dialog box Figure 5 4 www AccuriCytometers com Page 43 Rename Parameters Rename FLi to Fire FL1 Select Rename FL2 to JeD1 1b PE FL2 Salen CO3 FITC Rename FL3 to PE C FLZ eee Rename FL4 to are IFL41 FITE Apply bo f Sample HPB CO11b PE OK Cancel i All samples Figure 5 4 Rename Parameters selection options menu The Display Menu F4 untitled Workspace CFlowm File Edit Display Instrument About Events Display Settings Remove All virtualsain Calculate Median Ctrl F Recalculate All Medians 1 2 3 4 3 6 8 9 10 11 12 at a as aa as AB A7 ABl AD A10 A11 A12 Bi B2 BS B4 B85 BG BF BS B9 B10 B11 Biz G1 f Gz f Cs p C4 Ch CGR Ce PCa CS PCIOPCTp CZ D1 D2 Bs D4 DS DE D7 DS BS D10 D11 Diz E1 ELF E3 E ESF ECP E7 ES E9 E10 E11 E1 Fig Fg FaFa FSFE pF p FS p FS Ge Ue gF i G1 G2 Ga G64 65 Gh GF GB Ga G10 611 G12 too Tm Oooo P H1 H2 HS H4 HS HE H7 HS He H10 H11 H1 Figure 5 5 The Display Menu Events Display Settings Used to change the number of events displayed in all plots There are
23. Gate Mo Gating Gate Mo Gating 219 048 Figure 2 1a 100 000 S5C 4 200 000 400 000 600 000 300 000 SSC A sid ddd 1 000 000 1 600 000 FSC A Gate F1 in all z Gate P1 in all pa ui i af Figure 2 1b 4 aot Fr CDS FITC A a CD3 FITC A ac als an im wl ae ae t ae t av act a i CD45 FE CY7 A CD45 PE Cy A Figure 2 1 Zoom tool used to focus on lymphocyte light scatter and surface marker fluorescence Fluorescence compensation can be set during or after data collection Data collection on the C6 is digital This means that fluorescence compensation used to adjust the spectral overlap from one fluorochrome to another can be applied or adjusted even after data has been collected and saved in a CFlow file Each data well in holds a maximum of 1 million events Data can be added to a well even one already containing events at any time up to a total of 1 million events When a data well already contains data the well will be colored blue and the RUN bution will read ADD TO www AccuriCytometers com Page 16 Chapter 3 Using the Collect Tab Introduction The Collect tab appears when CFlow is launched and is the only tab which allows control of the C6 The Collect tab is used to set data collection thresholds and gates stop criteria and to control the fluidics including sample collection using the RUN and PAUSE buttons Collect also allows access to most of the analytic functions i
24. H H8 HO H10 H11 H12 w CD45 PE Cy7 A s 200 000 400 000 590 477 FSC A Jope sae Plot 6C Sample HPB CD45 PE Cy 7 Count Volume uL of This Plot of All Mean FL1 A MeanFL4A CVFL1 A CVFL4A_ Median FL1 A Figure 4 6 Analyze Tab Row 2 selected to display data from Sample A2 in Plots 1C 3C and 6C 11 Use the Scroll Bar on the right hand side of the Analyze tab Workspace to scroll down bringing six more plot corrals into view Figure 4 7 PB 4 Color Tutorial Analyze CFlow Edit Display Instrument About A accurl Analyze A2 HPB CD45 PE Cy 7 123 4 5 6 7 8 9 10 11 12 EKATE AB A7 AB AD AIO A11 A12 B1 B2 B3 B4 B5 B6 BF BS B9 B10 B11 B12 C11 c28 C3 c44 c51 cef cry cs cg c10 C11 C12 Select a plot and Select a plot and Select a plot and sample to analyze sample to analyze sample to analyze D1 D2 D3 D4 DS DB DF Ds D9 D10 D11 D12 E1 2 E3 E4 E5 E6 EF E8 E9 E10 E11 E12 F4 F2 F3 F4 FS FO FF F8 FO FIO F11 F12 61 62 gt 63 64 G5 GB G7 G8 69 61016111612 acy leu Seb tng fe feel eel bos H1 H2 H3 H4 HS H6 HF HS HG H10 H11 H12 Make a new plot Copy Pits trom Colect 4al sl al ha Plot List Plot 1C Fsc a ssc a Plot 2C FL1 4 FL2 4 Plot 3C FL1 4 FL3 4 Plot 6C FL1 4 FL4 4 Scroll Bar Select a plot and Select a plot and ECT a plot and sample to analyze sample to analyze sample to analyze Plat AA Gamnle HPR CN45_PF_Cw 7
25. Histogram in Analyze Tab cccccccccccssseeeeeeceeeeeeeeeeeeeeeeeeeeeeseeeeseeaeeeesessaeeeeeesaageeeeees 41 Chapter 5 CFlow Software Menus ccccccccccsssseeeceeeeeeeaeeseeeceeeeeeeaeeesseceeeeesseeeaseeeeeeesssseeasseeeeeeeesaaaeeeeeeess 42 TEFEN e a E E A cima nacnnseuen E 42 TEE OLN ONU eea E E E E EA 43 TAE ADOU MENU resne tes dees sec sasence es adesee Gee tse st ageencee aeaeteeaser aes oe nea teoet wees osate lt ecteadcasonenactecssueco eens 48 GION WMG ell Gre cassette ates cate oe eptaetnaceveeeeigo E E 49 Applying WE 2 UA rset acces sceteren cen cecal EE AE EEEE 50 CIR OFOW INO e e ence suena aac cenetn Genus seieetcne soee aucun seen see E E E 56 PP AIO GUNG saat ohare saa T oteneeoa ne suvencteacsacdaearesesasevecostanercencauaionoaaas 56 Wie TATA OMS CU AG aoe ste stese E sects E E A 58 Using the Manual Collect Tab ccccccccsscseccccceeceeeeeseeeeeeeeeseeeseeceeeesseeasasseeeeeessaeaaseeeeeeeessaeaaeeeeeesssaaaaasss 60 Pe Ano SOM CCL WA see aie recs E EE E TEE O 61 Creating Sample Sets and Applying Acquisition Settings ccccccccssseeeeeeeeeeeeeeeeeeeeeeeeeeaeenseeeeeeessaaaees 62 TAE RUN DI PII y ee E 65 Analyze and Statistics VAD Son siccavencsicnsas5 insana nn oaeee rta sebaeceneaecsacavaceeupasaug NEANS ERRANEN SENNA 67 Running a Sample Plate Example cccccccccccceceeceeeeeeeeeeeseeeeeeeeeeeeeessseeeseeeeeeesssseaseeeeeessssaasesseeeeeess 68 Collisions and Alignment asrasin a a ania
26. Option iS CHOSEN ssurersisirin mass 26 Figure 3 19 Saving and naming a CFIOW Fil ccccccccccccccceeeseeeeeeeeeeeeeeeeeeeeesseaeseeeeeeeeeseeeaaeeeeeeesaeaaeasses 26 Figure 3 20 New file name Human PB appears in the upper left corner of the CFlow workspace after saving and naming the CFIOW file ce ecccccceccsssssseeceeeeeeeaeesseeeeeeeeeeesseeceeeeesseeeaseceeeeesseaseeeeeeeeeessaaageeeeeeees 27 Figure 3 21 Fluorescence spillover of positive PE Cy7 signal from FL3 into a FL1 A and b FL2 A c The PE Cy7 signal does not spillover into FL4 A ccccccccccccccssseeeeeeeeeeeeeeeeeeeeseeeeseeeeeeeesseaaeeeesseaaeeeeeessaeeeeeeesas 27 Figure 3 22 Selection and placement of Quadrant Marker cccccccccsseeeeeeeceeeeeeeeseeeeeeeeesaaeeeeeesaaseeenees 27 Figure 3 23 a Placement of Quadrant Tool for Median calculations on the double negative lower left and single PE Cy7 positive upper left populations b Median fluorescence values in the Statistics Table for Plot 3 Q1 will all read NA until Calculate Median is selected cccccccssseeeeeeeeeeeeeeeeeeeeeeeeesaaaeeeees 28 Figure 3 24 Quadrant Q2 is selected with mouse click indicated by bold red and Calculate Median chosen from the Display drop down MENU ccccsssseccccceeeececceeseeceeccauseeeecceuauseeeessuaueeeessaaseeeeessaageeessssaaass 28 Figure 3 25 Statistics Table updated with median values after choosing Calculate Median
27. R and Marker M gating regions that have been drawn will appear automatically in the Gating dialog box list of options To use Vertical or Quadrant markers for gating select the appropriate check box in the Gating dialog box and those choices will appear Figure 3 13 Change Gating for Plot 2 xa Gating choices E Include ay ee ji i Vertical and Quadrant Marker Check Boxes Clear All Apply Close Figure 3 13 Adding Vertical or Quadrant Markers to the Gating list The initial display of any parameter on a logarithmic scale will be automatically zoomed to a scale that runs from channel 10 to 16 7 x10 For the majority of analyses there are very few events which will fall into channels O to 10 Pre zooming of the plots will save time by reducing the number of zoom steps However care must be taken when setting Markers M Regions R or Polygons P that require inclusion of channels lower than 10 on a pre zoomed plot It is recommended that users unhide the first decade of data especially when setting fluorescence compensation as this will prevent events from being excluded from gating regions Refer to Figure 3 14a and b for directions www AccuriCytometers com Page 23 Plot 2 Sample Unstained GATE F1 in all EREIN X AXIS FL1 W C linear FLi 4 W linear Min Yalue Max Value log Min Value Max Yale log Hide 1st 10 sift 777 215 M eel Hide 1st E decade Range 10 16 777 21
28. The CSampler is an optional accessory for the C6 flow cytometer that allows for the aspiration of samples prepared in 48 and 96 well plates and standard 12 x 75 mm tubes from a 24 tube rack In order to operate the CSampler CFlow Plus software must be upgraded to CFlow Sampler software CFlow Sampler is based on the CFlow Plus platform so much of the functionality is common to both platforms This chapter outlines the additional capabilities of CFlow Sampler and differences from CFlow Plus Increased Data Capacity CFlow Sampler has a higher total data capacity than CFlow Plus When a 96 well plate is selected the total event capacity has been increased to 96 million events Each sample well is still limited to 1 000 000 events The total event capacity has been similarly increased to 48 million events for a 48 well plate and 24 million events when utilizing the 24 tube rack Data Collection Tabs CFlow Sampler has two tabs that can be used during data collection Manual Collect and Auto Collect The Manual Collect tab is designed for running individual samples from a plate or tube rack It can also be used to define the parameters which are going to be used to acquire data prior to data collection for either collection mode The Manual Collect tab is also used to re collect data from wells that have been previously run from the Auto Collect tab The Auto Collect tab is designed to automatically collect data from multiple samples in microplate
29. a a enaa a aaa a a aaa 69 Table of Figures Figure 1 1 The CFlow Control Panel in detail ccccccccccccccssseeeeeeeeeeeeeeeseeeeeeessaeeaeseeeeeeesseeeaeeeeeeeeessaaaseses 5 Figure 1 2 C6 Improper Shut Down Recovery Message cccccccceeceeeececeeeeeeeeeeeeeeeaaaeaaaaaaaaaeeeeeeeeeeseesaaas 6 Figure 1 3 Saving a CFIOW file cccccccceeccceeeceeeesseceeeeeeeeeeeeseeeeeeeeesseeeaeceeeesseeesseeeeeeessseeessaseeeeeeesssaaaaeses 8 Figure 1 4 Proper position of R1 on Plot 1 and Gate selection for Plot 2 0 0 0 cceceeccecceeseeeseeeeeeeeeeeeeeseeeeees 9 Figure 1 5 Location of Zoom Tool and Expand Tool and effect Of ZOOM cccecccecseeeeeeeeeeeeeeeseeeseeeeeens 9 Figure 1 6 Example of 8 peak bead data collected with a C6 The number of expected peaks is FL1 H 8 FL2 H 8 and FL3 H 6 The peaks in FL4 H are not relevant cccccccseeceeeeeeeeeeeesaeeeeeeeeeaeeeeeeeeas 10 Figure 1 7 Example of 6 peak SPHERO APC Calibration Particles shown in FL4 H The peaks in FL1 H FL2 H and FL3 H are not relevant cccccccccccesccceesceceeeeeceneeececeeseueeseaeeessaeeesaueesseeeseueeesueessuseeessneeesagees 11 Figure 1 8 Statistics tab Display of 8 peak bead data from SUCCESSIVE CaYS cccceceeeeseeeeeeeeeeeeeeeneeees 12 Figure 2 1 Zoom tool used to focus on lymphocyte light scatter and surface marker fluorescence 16 www AccuriCytometers com Page 1 Figure 3 1 CFlow Workspace after selecting
30. an individual sample Apply Settings After a sample or set of samples has been selected input the criteria by which these samples are to be collected This includes Run Limits Fluidic settings Slow Med Fast and the desired threshold s The option for custom fluidic settings Flow Rate and Core Size has been disabled in the Auto Collect tab Wash cycles 0 5 can be chosen to occur after each sample well is complete Apply settings by clicking the Apply Settings button Selected samples will be highlighted the same color once settings have been applied to the set or group of samples Figure 7 9 www AccuriCytometers com Page 62 If Run Limit or Flow Rate and Core Size settings are modified using the Manual Collect tab those settings will not change in the Auto Collect tab Threshold settings and Color Compensation settings will apply to both tabs 2 Sampler Test Save CFlow File Edit Display Instrument About 2 Untitled Workspace CFlow File Edit Display Instrument About Cfrlow sampler Cflow sSampler Auto Collect Auto Collect 5 W Plate Type 96 well plate Eject Plate Plate Type 36 well plate Eject Plate ex A Plate Name Plate Name a A Select All A l l Select All A Ctrl click to view sample settings Deselect All Ctrl click to view sample settings Deselect All m 1 2 3 4 5l6l 7 89
31. ato att A12 B1 B2 B3 f B4 f BS f BG BF BS Plot 1 Sample 8 Peak Be GATE No Gating Plot 2 Sample 8 Peak Be GATE R1inall Plot 3 Sample 8 Peak Be GATE R1 in all R1 87 5 A B9 B10 B11 B12 C1 f c2 f C3 c4 cs cop c7 cs co C10 C11 C12 i i D1 f D2 D3 D4 DS DG D7 D oO D9 D10 D11 D12 6 et E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 F4 F2 F3 F4 FS FG FF FS FO FIO F11 F12 2 2 5 a ol a w t D 2 FL2 H 61 621631641651 G66 67 GB 69161016111612 E a Tm eS gp H1 H2 H3 H4 HS HG HF HSB H9 H10 H11 H12 oes A E Plot 6 Sample 8 Peak Be GATE R2in all d C6 and CFlow are connected H Plot 4 Sample 8 Peak Be GATE R1 in all Plot 5 Sample 8 Peak Be GATE No Gating Run Limits i JZ 50000 events fio min fo sec E boo pL Backflush Unclog Fluidics Slow Medium Fast Flow Rate 14 pL min Core Size 10 pm C Custom R2 50 1 Flow Rate 14 pL min M4 p 0 at Pag 2 FL4H z Ot Threshold Set Threshold 80 000 on FSC H None wl at aed JL Plot 1 Sample 8 Peak Beads 2 9 09 Count Volume pL of This Plot of All MeanFSC H Mean SSC H CVFSC H CVSSC H Median F Set Color Compensation F All 50 000 46 6 100 0 100 0 560 132 7 _ 580 097 2 11 7 27 3 n x umulative Delete Sample Data Ril 43 747 46 6 87 5 87 5 549
32. events from the pop up gating list 4 Position the predrawn horizontal Marker Region around the top brightest far right peak on each of the 3 fluorescence plots Use the Zoom Tool located in the lower right corner of each plot the button with the magnifying glass to zoom in on the top peak Then adjust the marker tightly around the peak Click on the Expand Tool the button with a square and four arrows to zoom back out 5 Compare the bead run to the manufacturing results sent with the C6 If the C6 is performing properly data should look similar to that in Figure 1 6 for the 8 peak beads There should be one main www AccuriCytometers com Page 8 population of beads on FSC H vs SSC H 8 discernable peaks on FL1 H and FL2 H and 6 peaks on FL3 H FL4 H performance will be checked with the 6 peak beads in a later step Plot 1 Sample 8 Peak 103 Bd E Plot 2 Sample 8 Peak 103 GATE No Gating Ei GATE R1inall F D Gate Applied Plot 2 Sample 8 Peak 103 i Plot 2 Sample 8 Peak 103 GATE R4 in all ue GATE R4 inall Zoom Tool Expand Tool A Before Zoom B After Zoom Figure 1 5 Location of Zoom Tool and Expand Tool and effect of Zoom www AccuriCytometers com Page 9 Daily Bead Analysis from Bead Template 1727 CFlow ei nw File Edit Display Instrument About Clow Plus Collect A1 8 Peak Beads 2 9 09 13 2 3 4 5 6 7 8 9 10 11 12 E a as aa as asl a7 as ao
33. events FSC A SSC A ne Select this option to Gate Plot 2 on all FREA FLA events falling in P1 goe S88 Clear All Apply Select i Tyne Select ne alart ri E IV A CICO DIOT LVA G wlil DIOT tVbe WEeIiCCl DIOT tVDe Figure 3 10 Selecting a gating option which includes all events from a given gate i e Plot 2 is now gated to Include P1 on all events Close 5 Apply a gate to the FL1 A vs FL2 A plot Click on the gray GATE button at the top of the plot to open the gating dialog box Figure 3 10 6 Select the gating option Include P1 on all events in the Gate dialog box and click Apply at the bottom of the box The resulting gated Plot 2 is shown in Figure 3 11 Note that the text next to the gray Gate button will now read P1 in all instead of No Gating www AccuriCytometers com Page 22 Plot 1 Sample Unstained Plot 2 Sample Unstained GATE No Gating GATED P1 in all me Figure 3 11 Plot 2 has been gated to include P1 all events The events within any gate or series of nested gates can be selected to be included or excluded from a plot In addition gating combinations that do not appear as options in the Gating dialog box can be created by selecting the intersection choice Figure 3 12 Change Gating for Plot 2 Gating choices Include Exclude H Intersection Figure 3 12 Gating choice options for plots Include Exclude and Intersection Only Polygon P Rectilinear
34. is started up again after this cycle a longer fluidics Startup cycle will run and CFlow will indicate that an improper shut down has occurred This longer cycle will purge cleaning reagent from the flow cell and the C6 will be ready to use when Startup is complete CAUTION Never run the Clean Flow Cell cycle without a tube containing at least 250 uL of fluid on the SIP Calibrate Fluidics Initiates the fluidics calibration feature of the C6 which will ensure that the instrument provides accurate measurement of the volume aspirated from samples Update Firmware Updates the C6 firmware Use only when directed by Accuri Cytometers to upgrade the C6 firmware with an official firmware release The About Menu EJ untitled Workspace CFlowm File Edit Display Instrument About Clow Plu About CFlow Technical Support Information Get a6 internal log wE i ooo O E4 F4 a1 rcoa7T7 mr ono p H4 Figure 5 14 The About Menu C2 De Ez Fiz 2 Hz CS D3 ES Fis ve H gt 4 H4 ES F5 Go HS EG Fei 6 HE EF Fy i HF Le DS ES FS Hg 10 11 12 AIO A11 AZ B10 B11 Biz Cia C11 C12 D10 D11 B12 E10 E11 E12 FAQ F141 Fiz 10 G11 1z H10 H11 H12 About CFlow Opens a dialogue box containing the version of CFlow currently installed It also has the contact information for Accuri Technical Support Technical Suppor
35. least 2 mL filtered deionized H O 5 Firmly press the On Off button on the front of the unit to turn on the C6 A blue light will flash and then stay illuminated After 20 seconds the CFlow traffic light will show yellow After 60 seconds the C6 pumps will start to run When powered on the C6 will automatically flush the fluidics lines with sheath This process takes approximately 3 minutes 6 When the C6 is ready for data collection the CFlow traffic light will change to green Begin data www AccuriCytometers com Page 5 collection when the Traffic Light is green and the C6 Status Message Box reads C6 and CFlow are connected and ready For optimal performance the C6 should be on for at least 15 minutes prior to processing samples C6 Improper Shut Down Recovery Message If the message shown in Figure 1 2 is seen at startup the C6 will take approximately 6 minutes to recover and return to the green light ready state as opposed to the usual 3 minute startup procedure This may occur on initial C6 startup when the unit is first received It will also occur after an interruption of power to the unit H1 H2 HJ Ha H H H7 H H4 H10H11 H12 gi CE was improperly shut down wf Resetting for normal operation Figure 1 2 C6 Improper Shut Down Recovery Message Running Validation Beads Accuri provides an initial set of pre diluted validation beads Store the beads at 4 C in the dark The beads expire within 2 weeks of rec
36. pm C Custom fo Min J0 Sec Flow Rate 23 pLimin feo ow eet Core Size J Core Size 10 pm Wash and Backflush _Unclog_ Threshold Aaitate Controls Wash Agitate Set Threshold 80 000 on FSC H None Set Color Compensation Last Run Cumulative Delete Sample Data 0 Events 0 0 00 Time 0 00 V Warn before deleting 0 0 Microliters 0 0 0 Events Sec 0 Data Capacity Used 0 0 Events uL 00 o of 96 000 000 Events Figure 7 3 The CFlowSampler Manual Collect Tab There are several unique features in the Manual Collect tab the ability to load and eject a plate the option to wash the SIP between samples and the ability to agitate or mix samples Load Eject Selecting the Load Eject function will move the CSampler arm and plate platform in or out of the C6 to facilitate the placement of a plate or tube rack on the plate platform or remove a plate after sample acquisition is complete Figure 7 4 After 15 minutes in the eject position the arm and plate platform will automatically return to the home position with the SIP in the wash station Wash Clicking the Wash button will activate a single wash cycle thoroughly cleaning the SIP of any previous www AccuriCytometers com Page 58 sample The contents of the SIP are aspirated up and out of the SIP then the SIP and wash station are rinsed with clean sheath fluid Selecting Wash from the Manual Collect tab will perform a single wash cycle Using Wash from the Auto Collect Tab all
37. replacement procedures and Section 6 for order information Keep the C6 Validated Run the Accuri recommended Validation Beads each day the C6 is used Refer to Chapter 1 for detailed instructions on Validation Beads If CFlow Plus is installed on the C6 and volumetric or absolute cell counts are required it is important that the system is accurately measuring the volume aspirated from each sample o Replace the peristaltic pump tubing every 2 months o Ensure that the SIP and flow cell are clean and the fluidics lines are free of air bubbles Refer to C6 Flow Cytometer Instrument Manual Section 5 46 Running the Cleaning Cycle and 5 5 Inspection of the Fluidics Lines for directions o Validate volume measurement using Accuri s Volumetric Validation Beads or other counting bead product Refer to C6 Flow Cytometer Instrument Manual www AccuriCytometers com Page 13 Standard Operating Procedures for the C6 The following are recommended standard operating procedures Instrument Startup 1 Check fluid levels in all tanks and attend to them if needed 2 Press the Power Button on the front of the C6 and open CFlow Software 3 Wait until the Traffic Light in CFlow is green and message reads C6 and CFlow are connected and ready Place an empty 12x75 mm tube on the SIP Click BACKFLUSH button in CFlow A small amount of sheath fluid should appear in the tube Place a fresh tube containing 2 mL of filtered deionized H20 on t
38. the negative population Figure 3 29 blue boxed values 12 Select Apply to All samples then the Save amp Close option in the Compensation Settings dialog box in order to apply these Color Compensation settings 13 The FL3 PE Cy7 signal does not spillover into the FL4 channel significantly as indicated by comparison of the Median FL4 fluorescence channel numbers Figure 3 30 Therefore there is no need to subtract FL3 signal from FL4 14 The next sample collected is number 3 stained with CD45 PE Cy7 and CD3 FITC Table 3 1 There is significant spillover from the FL1 FITC signal into the FL2 channel Figure 3 31a riot 1 Sampie a riot lt ampie ss XT GATE No Gating FIOT 5 Sample He E l Compensation Settings for Sample A2 Es a Correct FL1 by subtracting a percentage of Fiz P Yo alse UE Yo FL4 000 Correct FL2 by subtracting a percentage of FEIS F Yo lsh s 30 Yo FL4 F Yo 209 000 1 U 0 250 Correct FL3 by subtracting a percentage of Choose an option FL1 F Yo FL2 c Yo FL4 F A here Correct FL4 by subtracting a percentage of ELI P Yo Fz P Yo FL3 P Yo Plot 4 Sample A2 Apply to Choose this option to bad ese Preview tee e Save Compensation All samples Settings and close the dialog box wt S w CD45 PE Cy7 A we of All Mean FL2 A Mean CD45 PE Cy7 A CV CD4
39. this sample Apply Close C All samples Figure 5 12 Warning displayed after a change in threshold settings 30 OPTIONAL SECO ngate Permanently el Set Compensation Opens the Compensation Settings dialogue box The Compensation Settings dialog box can also be accessed from the instrument settings panel of the Collect tab under the Run button Click on the parameter to be subtracted in order to enter a percentage subtraction value In the example shown in Figure 5 13 the FL2 button in the upper left corner has been clicked and the numbers in the percent box appear on a white background A subtraction percentage value may now be entered A grayed out box is not accessible until the parameter button to its left is clicked Compensation Settings for Sample A1 X Correct FL1 by subtracting a percentage of 250 FL2 0 00 FL3 I 00 Flg Po Correct FL2 by subtracting a percentage of Fu p o Fis o oo ne Correct FL3 by subtracting a percentage of Pi P FL2 I 00 FL4 10 Correct FL4 by subtracting a percentage of Apply to e kat 2 Save amp Cancel amp 3 ja Preview Close Close All samples Figure 5 13 Compensations Settings Compensation can be applied to the currently viewed sample or all samples simultaneously by choosing one of the options under the words Apply to in the lower left hand corner The Compensation dialog box can remain open and in view thro
40. 11 B12 1 Overlay Tool 414 042 D1 b11 D12 E1 2 E3 f E4 ES f E6 EF ES EX E10 E11 E12 D F41 f F2 gt F3 F4 FS FG FF FS FQ O F141 F12 61 62 63 64 65 GB G7 GB GO G10 G11 G12 coomTmTmmond wo p H1 H2 H3 H4 HS HG H7 HS HS H10 HN H12 POTE Mite Make a new Copy Pits tromColect al if a bl Plot List Plot 1C Fsc a ssc aA Plot 2C FL1 4 FL2 4 Plot 3C FL1 4 FL3 4 Plot 6C FL1 4 FL4 4 Plot 8 FSc A Figure 4 10 Analyze tab Overlay Tool has been selected Analyze eee S BD R1 in P1 in al d verlay Histogram Legend 123 4 5 6 7 8 9 10 11 12 a hh hi vs AB A7 AS AD AID A11 A12 B10 B11 B12 10 000 12 8 B1 B2 B3 B4 BS BG BF BS B o Hi Sample HPB CD45 PE Cy 7 Hi Sample HPB CD3 CD45 Hi Sample HPB CD3 CD4 CD45 CD8 C1 c2 C3 C4 CS C6 CF csi c9 010 011 C12 D1 D2 D3 D4 DS DEB DF DS D o B10 B11 B12 E1 E2 E3 E 4 5 E6 EF E 8 E o E10 E11 E12 F1 E F2 F3 F4 FS FG FF FS FO FIO F11 F12 G61 G2 G3 64 G5 G6 G7 G8 G9 G10 611 612 ae Om m oO O p wot gf a 2 H1 H2 H3 H4 HS HB H7 HS H9 H10 H11 H12 FLA Multiple names Make a new plot a Copy Plots from Collect Aa p jl o bal H 5 s A Plot List Overlay Legend Tool Figure 4 11 Analyze tab Overlays of FL1 A data for samples A2 A3 and A4 The Overlay Legend has been opened Plot 8 FL1 4 6 Selecting the Overlay Histogram Legend Tool opens t
41. 3 options display all events acquired display the first specified number of events in the data file or display a percentage of the whole If percentage is chosen it will be a pseudo random selection For example if 20 is selected every 5 event will be displayed C Display First 50000 events collected C Display 2o percent of events collected Apply OK Cancel Figure 5 6 Events Display Settings Remove All VirtualGain Removes all VirtualGain settings that have been applied from the entire CFlow workspace VirtualGain settings will be removed from all wells of data Refer to the VirtualGain chapter for more information www AccuriCytometers com Page 44 Calculate Median Before selecting this option the marker or region where the median is required should be made bold by clicking on it with the mouse Figure 5 7b If a median has never been calculated for a marker or region the notation NA will appear in the Statistics Table Figure 5 8a Once the Calculate Median option has been selected the result will be displayed in the statistics pane of the Collect or Analyze tabs Figure 5 8b i Gate P1 in all A Gate P1 in all ai 2 a E E E E 3 n ae ae 4 t k k wI afl ia CD3 FITC A CD3 FITC A Figure 5 7 a Quadrant marker is not selected b Quadrant Marker selected bold lines p a Figure 5 8 a Statistics table before selecting Calculate Median b Statistics table after selecting Calcu
42. 5 Set Plot Specs for Plot 2 x X AXIS linear Plot 2 Sample Unstained GATE gt P1 in all Set Plot Specs for Plot 2 xj Uncheck Hide 1 decade 0 16 777 215 linear Min Yalue Max value log 10 16 777 215 Vie Range 10 16 777 218 Apply OK Cancel Hide 1st a decade 0 16 777 215 Range 0 16 777 215 OK Cancel Figure 3 14 Selecting to display the 1st decade of data on a default log data plot a Open the Plot Spec dialog box b Uncheck Hide 1st decade boxes for FL1 A and FL2 A and set the Min Value for both parameters to 0 7 Add 4 more fluorescence density plots and apply the P1 gate to each For this analysis the plots shown in Figure 3 15 will be used to set appropriate fluorescence compensation IHD Unatined Al Crow Eas File Edit Display instrument About Crlow Plus Collect Plat I Gale Ue Pict t temple Unna os GATED Min ail ia GATED Ho dmrg Al unstained ts 123 4 5 amp fF 10 11 13 FE azo aa aa ao a aa ato ant tz Bi B2 i BS B bS B E7 Ba BTO Bt Baz 2 3 04 C8 Of cr Co Oho Chl One 28 8 es 2i afi ps bo OF i et 2 Fo Fa Pe Pe Pe aa a eae Se ee Pe ae rar 6S P s b Et Ef EX E1 ES E0 EF EE E Br etl Bae F F Hi TEN MIER T EE TE EN TEN
43. 5 PE Cy7 A Median CD45 PE Cy7 A 42 578 83 NA 96 751 0 0 95 2 42 0 147 1 44 694 0 86 8 AS EE 45 395 0 1 0 0 0 0 0 0 10 066 0 62 966 0 0 0 0 0 0 066 0 62 966 0 4 908 0 0 4 8 2 1 147 4 877 5 84 0 EET 767 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 oa Figure 3 29 Results of correcting FL2 by subtracting 4 3 of the signal from FL3 PE Cy7 The Median FL2 fluorescence channel value for Q2 UL 131 is approximately equal to the Median FL2 fluorescence channel value for Q2 LL 134 Plot 5 Sample 42 Plot 6 Sample A2 GATE P1 in all GATE P1 in all z w S d 3 3 Oo ll Mean FLA Mean CD45 PE Cy7 A CV FLAA CV CD45PE Cy7 A Median FLA Median CD45 PE Cy7 A a a eh A 2 340 5 42 578 8 93 1 35 8 NA NA Vo 342 4 44 698 8 93 3 27 6 290 0 45 395 0 l 0 0 0 0 0 0 0 0 0 0 0 0 to 304 0 884 4 87 9 70 6 263 0 771 0 Vo 0 0 0 0 0 0 0 0 0 0 p Figure 3 30 No fluorescence subtraction of FL3 PE Cy7 signal is needed from FL4 as indicated by comparison of Median FL4 values for Q3 UL 290 and Q3 LL 263 www AccuriCytometers com Page 31 k Gate F1 in alli i Gate F1 in all ut i a FLZ A w COs FITC A COS FITC A Spillover of FITC a positives b Figure 3 31 Correction of spillover from FITC FL1 into FL2 a Before fluorescence subtraction b After calculation and application of appropriate percentage subtraction of FL1 from FL2 15 Correct the spill of the FIT
44. 64 loz oa oa s 0 07 68 68 610 c1 612 Jammas oeg E ai ese a ai misa7 O H1 H2 H3 H4 HS H6 HT He He H10 H11 H12 4 J 4 TrM nn wo gt Make a new plot Copy Plots fromCollect Aal l bal i Plot List Plot 1C FSC A SSC A Select Plot 2C FL1 A FL2 A Plot 3C FL1 A FL3 A Plot 6C FL1 A FL4 A Select a plot and Select a plot and Select a plot and sample to analyze sample to analyze sample to analyze Figure 4 3 Plots 1C 3C and 6C opened in the top row of Analyze tab www AccuriCytometers com Page 37 ACCU Collect Analyze Statistics A1 Unstained Plot 1C Sample Unstained Plot 3C Sample Unstained a Plot 6C Sample Unstained f AGATE No Gating AGATE No Gating e GATED No Gating A B C D E F G st 62 63 G4 os o6 67 os oe o10 o11 012 0 200 000 400 000 590 477 H Ht H2 na Hal He He H7 e Ho H10 H11 HZ PSCA ie oF fe gt a H gop sae a piotsromcoiect 4al sl l bal rel gog Bak Plot List Plot 1C FS5C A 55C A Plot 2C FL1 A FL2 A Plot 3C FL1 A FL3 A Plot 6C FL1 A FL4 A Select a plot and Select a plot and Select a plot and Figure 4 4 Analyze tab with copied plots in Plot List and selected plot corral Flow ACCU Collect GATE P2in all GATED R2 in P2 in al A1 Unstained Plot 1C Sample Unstained a X Plot 3C Sample Unstained a X Plot 6C Sample Unstained X i GATE gt
45. 75 mm tube on the SIP Click on BACKFLUSH Place a fresh tube with 2 mL filtered deionized H20 on the SIP Click on well A1 to select it Well A1 should now have a red box around it i OY p ee Deselect the Run Unlimited check box under Run Limits in the Instrument Control Panel www AccuriCytometers com Page 6 9 Select the time check box Min Sec in the Instrument Control Panel and input 2 minutes 10 In the Control Fluidics section of the Control Panel select Slow The flow rate should display 14uL min and Core Size should display 10 um 11 Click on the Threshold button to set the Threshold to 80 000 on FSC H default 12 Press Run While data is being acquired well A1 will flash blue 13 Click Pause then the Delete Sample Data button in the Control Panel of the CFlow screen to clear data collected during the run The data well should change from blue to white and the counters will reset 14 Deselect the time check box and click the Events check box Verify that it is set to 50 000 events in Ungated Sample If not enter 50 000 events and select the Ungated Sample check box 15 Replace the tube on the SIP with a tube containing suspended 8 peak beads Vortex the tube prior to placing it on the C6 16 Click RUN to start acquisition Acquisition will automatically stop after 50 000 total events are acquired Do not click on ADD TO ADD TO is used to add more data to a well that already contains data The R1 region m
46. A2 data to display in each plot Figure 4 6 The gates set on Plots 3C and 6C in Steps 8 and 9 are applied automatically to the appropriate plots in Row 2 File Edit Display Instrument About S J0 FI I2 Ma a ma a AB r rar os ag A10 A11 a12 B1 EI B3 B4 BS es 87 Be B9 B10 B11 B12 eee Select a plot and Select a plot and sample to analyze sample to analyze C10 c11 c12 P p10 D11 biz Select Check Boxes 61 62 esl cal 65 csl ezl ca Plot 1 FSC A SSC A I Plot 2 FL1 A FL2 A H1 H2 H2 H4 HS H6 H7 HS Make a new plot Copy Plots from Collect J Au a bal J Plot 3 FL1 A FL3 A J Plot 4 FL2 A FL3 A Plot List To add plots make a new plot TT Piot S FL Ari3 A or copy from Collect J Plot 6 FL1 A FL4 A Note Gating information will not be copied OK Cancel Figure 4 2 Selecting plots to copy from the Collect tab ec Analyze No Sample 2 93 4 aw oF Z 9 10 1 i 12 AB A7 ABU Ag Ato At AP At a12 Select a plot and Select a plot and Select a plot and sample to analyze sample to analyze sample to analyze g a E a a a aay j sia R D7 De D9 010011 D12 ajem joma ajo ais e2 E3 e4 E5 E6 E7 Ee E9 E10 E11 E12 E oS ag ae rte ei mn fiomnimniie mmea F1 f F2 f Faf Fa Fs Fe F7 Fe Fe F10 FA z a ye jo jomni jomnjoma o 3 e UR ar
47. C signal into FL2 by following steps analogous to 1 through 11 above using Plot 2 with the following differences e Apply a Quadrant marker and calculate the Median values for Plot 2 e Inthe Compensation Settings dialog box enter a value in the FL1 box under the line Correct FL2 by subtracting a percentage of Typically for any FITC conjugate this value will be between 6 5 and 7 5 e For this example the correct compensation value will result in approximately equal median fluorescence channel values for FL2 events in the lower left and lower right quadrants of Plot 2 Adding and Applying Additional Gates In this example CD45 will be used in addition to the light scatter gate to identify the CD3 CD4 and CD3 CD8 events This section will show how to use the Gate dialog box to create this nested gate 1 On Plot 3 CD3 FITC vs CD45 PE Cy7 select the rectilinear gating tool and draw a region R1 that encompasses the CD45 positive events Figure 3 32 Be sure to slide R1 off the left edge of Plot 3 to ensure that all events including those below channel 10 on the FITC axis are in R1 Flot 3 Sample 44 COS F GATE F1 in all Region R1 slid to left T 3 Rectilinear CD3 FITC A Gating Tool _ a EL Figure 3 32 Gating region R1 has been drawn on Plot 3 to include the CD45 PE Cy7 positive events 2 Click the gray Gate button above Plot 2 CD3 FITC vs FL2 A to open the Gate dialog box Figure 3 33
48. Count Valimefiul nf Thie Pint of All Mean FI 41 4 MeanFid A CWFI1 4 CWFI4LA Median FI 1 Figure 4 7 Analyze Tab view after scrolling down to empty plot corrals 12 Select Plots 1C 3C and 6C for these two rows of the Analyze tab and populate the plots with data from Sample A3 in Row 3 and Sample A4 in Row 4 Figure 4 8 www AccuriCytometers com Page 39 3 oe OV Elis accufi Ad HPE COS COs CD45 CDA Fat 1C Gample HFE OD ict 30 fanple HFE CD Moi ec iamma APE CI x L23456 70 9 0U aT Mo Gating i GATE Fz in w ATE JF in P2ing ata a afl ae AN AS ll A ADA ALS Bt BS Os esl ae ee ee are CV CS OF Oe oe Cope Ce OP emer at Ei Et EF EA ES ED EF EB Ep BIG E1 E12 a CH FE Gra t A oi oO Ge Bee Be ee eB AZ E F Fa Fa Fo Pa FE FS F7 FE Fo FID Fii FE G G1 GF ek tee GR Go Gig t 12 Ha Me H30 Haf ee ee l l a dS E Tio Baa Hor A TOE r Comet Piot irom Au fi a bal 7 i u F he Gt havea MPD CD GATED 22 iah iaw GAU 17 1 Pol BE FL hA FLA 614 Tit TT Teri ar h e af oft COF FT ce CEF FIP Ge Hoo Bae COL mae Figure 4 8 Analyze Tab view after adding 2 more rows of Plots 1C 3C and 6C and populating with data from Sample A3 and A4 13 To print directly from the Analyze tab click on the small white box in the uppe
49. D3 FITC vs FL2 A to include events in gates R1 and P ceeeeeeeees 33 Figure 3 34 Gate for Plot 2 CD3 FITC vs FL2 A includes events in gates R1 and P1 after following steps 1 3 in Adding and Applying Additional Gates ccccccccccccccssseeceeeeeeeseceeeseeeseceeeseeeeeeeeeseaaeeeeeeseaees 34 www AccuriCytometers com Page 2 Figure 3 35 Blue boxes show desired 4 log channel to view for Plots 2 ANd 6 cccccsseeeeeeeeeeeeeeneeeeees 34 Figure 3 36 Setting Min and Max channel values for the X and Y axes using the Plot Spec dialog box 35 Figure 3 37 Result of axes view adjustment to 4 logs for Plots 2 ANd 6 ccccccccsseeeeeeeeeaeeeeeeesaeeeeeeeeaas 35 Figure 4 1 The Analyze Tab ccccccssccccseseeecceseeeceeseeecsageccseeeeceeasecessaeeeesegseeeesagecessageeeeseuseesssaseeessageees 36 Figure 4 2 Selecting plots to copy from the Collect tab 20 2 ccccceceeeeeeeeeeeeeeeeeeeeeeeeeeeeeaaaaaeaaeeeeeeeeeeeeeeeeees 37 Figure 4 3 Plots 1C 3C and 6C opened in the top row of Analyze tab ccccccseeeeeeeeeeeeeeeeeeeeeeeeeeeaeeeees 37 Figure 4 4 Analyze tab with copied plots in Plot List and selected plot Corral cecceseeeeeeeeeeeeeeeeeeeees 38 Figure 4 5 Analyze Tab Result of gating Plot 3C on P2 in all and Plot 6C on R2 in P2 in all 38 Figure 4 6 Analyze Tab Row 2 selected to display data from Sample A2 in Plots 1C 3C and 6C 39 Figure 4 7 Analyz
50. Edt Display Instrument About CFlow Plus Figure 3 20 New file name Human PB appears in the upper left corner of the CFlow workspace after saving and naming the CFlow file 3 As more samples are run during this analysis session i e more data is added to this CFlow workspace the CFlow File named Human HPB will need to be re saved by selecting File Save CFlow File and overwriting the file This should be done often to ensure against loss of data Calculating Median Values and Applying Fluorescence Compensation A word about fluorescence spillover In this example some of the fluorescence signal from the CD45 PE Cy7 positive cells is picked up not only in the FL3 channel where it is expected but also in the unstained FL1 and FL2 channels This spillover is reflected in the shift to the right of the PE Cy7 positive cells from the upper left into the upper right quadrants of each plot in Figure 3 21 k Gate P1 in all a Gate P1 in all n Gate P1 in all 5 ai a A E E E No spillover of Spillover of Sf b positives a k Spillover of a 2 positives ah at positives at a m i es Backgroundof g Background of TRE T a negatives a s negatives T o4 wal Figure 3 21 Fluorescence spillover of positive PE Cy7 signal from FL3 into a FL1 A and b FL2 A c The PE Cy7 signal does not spillover into FL4 A This fluorescence spillover can be subtracted using a mathematical algorithm This pr
51. File New CFlow File from the CFlow file menu 18 Figure 3 2 The Plot Spec Dialog Box Changing these settings only affects the view of the data No data is changed in or lost from the data file ce ecccccceccccceeseceeeeeeeeeeeeceeceeeeeeseeeecesseeeeesseeeeeessaaeeeeseeeesseaeeeeeas 18 Figure 3 3 Threshold Settings dialog DOX cccccccssesseecceeeeeeeeeeeceeeeeeeaeeeeeeeeeeessseaeeeceeeeessaeaaseseeeeeeesssaaaaeses 19 Figure 3 4 Warning message when raising a threshold value will result in data lOSS eee 19 Figure 3 5 CFlow template after collection of Sample A1 human peripheral DIOO ccccesseeeeeeeeees 20 Figure 3 6 Use of Zoom Tool for a close up view of the lymphocyte population in a human peripheral DIOO SANDS METEEN dobwadeinedsidepasatennedexssnsbestedauebdemessadneetosnes 20 Figure 3 7 Polygonal gating tool was used to draw P1 on the 2D plot above Other available gating tools are Rectilinear and Quadrant cccccccsseseecccceeeceeeeeeeceeeeesueeseeceeeeeesaeeseeeeeeesseeaeseeeeeeessuaeaeeeeeeeeesessaaaeeseeeees 21 Figure 3 8 Opening a Density plot defaults to a linear FSC A vs SSC A plot ccecccsseeeeeeeeeeeeeeeeeeees 21 Figure 3 9 Changing density plot axes parameters and SCAIING ccseesceeeeeeeeseeeeeeeeeeeeeeaeeeeeeeseseeeeeeeeeas 22 Figure 3 10 Selecting a gating option which includes all events from a given gate i e Plot 2 is now gated to i
52. I Sample 42 All samples of This Plot of All Mean FL1 A 100 0 Sample HEE 000 FL4 0 00 FL2 O00 FL4 0 00 FL3 0 00 Save amp Mean CD45 PE Cy7 A P1 im all a2 Plot 3 Sample 2 GATE P1 in all CD45 PE Cy 7 A Plot 6 Sample A2 GATE P1 in all 2 Click Preview Button CV FL1 A Median CD45 PE Cy7 A 42 578 38 3 Compare 95 2 42 0 252 9 44 700 5 70 2 0 0 0 0 0 0 0 0 0 0 0 0 medians 0 0 T 2 1 269 9 887 2 87 6 0 0 0 0 0 0 7 973 0 1 145 5 27 8 Figure 3 27 Results of subtracting spillover of FL3 PE Cy7 signal from FL1 by 1 35 Note that the median FL1 channel numbers in Q1 UL 238 and Q1 LL 231 are approximately equal blue boxes Enter a subtraction percentage and click the Preview button in order to update the Statistics data When the appropriate subtraction percentage is reached the Median FL1 value in Q1 UL will be equal to that of FL1 in Q1 LL the negative population Figure 3 27 values in blue boxes Leaving the Compensation Settings dialog box open click on Plot 4 FL2 A vs CD45 PE Cy 7 set Quadrants as in Figure 3 28 and select Display Calculate Median from the drop down menu 200 000 FSC A Plot 4 Sample HPB CD45 GATE P1 in all Volume pL of This Plot 100 0 m 3 MW
53. No Gating A B C D E F a H Ht H2 H3 H4 HS HE OH He H9 H10 H11 H12 200 000 400 000 590 477 Make a new plot Copy Pits romcolect 4al sl al bal gogoe man Plot List Plot 1C FSc 4 sSc A Plot 2C FL1 4 FL2 4 Plot 3C FL1 A FL3 A Plot 6C FL1 4 FL4 4 Figure 4 5 Analyze Tab Result of gating Plot 3C on P2 in all and Plot 6C on R2 in P2 in all www AccuriCytometers com Page 38 CFlow Plus accuri Analyze A2 HPB CD45 PE Cy 7 Plot 1C Sample Unstained Plot 3C Sample Unstained Y Plot 6C Sample Unstained No Gati P2 in all R2 in P2 in al roon Gee anane D No Catina a ae P2 in all a ED R2 in Pein at ona a AS AB A7 AB AQ A10 A11 A12 B1 B2 B3 B4 B5 B6 B7 BS B9 B10 B11 B12 C1 f C2 f C3 C4 cS c6 c7 ces cg c10 C11 C12 D1 D2 D3 D4 DS DB DF DS DY B10 D011 D12 E1 2 E3 f E4 5 E6 EF f E8 E9 E10 E11 E12 Fi F2 F3 gt F4 FS FG FF FS FO FIO F114 F12 G61 G2 G63 G4 G5 GB G7 G8 69161016111612 Ir onmo 0w p 200 000 400 000 590 477 FSC A te py Plots fi Collect oO a i SPEC a nad r mij e oO Cc arec eq m i 0 rom Colle A al Al ae Plot 1C Sam A Sample HPB CD Plot 3C Sample HPB CD Plot 6C Sample HPB CD Plot List GATE No Gating GATE P2in all i GATED R2 in P2 in al 14 Plot 1C F5C A 55C A J Plot 2C FL1 A FL2 A Plot 3C FL1 4 FL3 A Plot 6C FL1 4 FL4 4 Hi H2 H3 H4 HS HG
54. Plus accuri Collect Pid 4 Garde Lire DARES Fo Gating 12345 6 Ff E 9 1011 2 A AI AJ AA ASU AD lU AT Aa A AID AIT A 7 BG Ba m0 eile 7 e ea SEDA Mt 200 eo y no 7 EG EG EW Bei i 5 D Dip ats Oa Db De caf D7 i pa po rinmi et E FR FE Fe Farad Faz E F i Fij Or OO o Oo 00 OT Ga Oo POTD H Bi Rat KO BA ee A A A 1D 8 bb aed Ce ane Gonnactad ad ree chy Saas O bedien amp Fe Fleer Fie 14 eine Com Site H pn i m 1 s LAL C Come Select plot type Zoom Tool Select plot type to make a new plot t to make a new plot Ad Ad Ce DOU 1h 4 re ee o Fumer 135 08 peat T ree aoa FF Warn Bator deleting GO Mbereditera r D Evii Sec D Dita Capitity itd G0 Ewerta fp 1 of 10 000 000 Ennis Figure 3 5 CFlow template after collection of Sample A1 human peripheral blood Creating Plots and Applying Gates Plot 1 Sample Unstained Plot 1 Sample Unstained GATE No Gating GATE No Gating z 2 Ss a S D a Ss a a D a a 3 a S a a zoom ui gt 0 500 000 1 000 000 1 600 000 FSC A gop gar h 200 000 485 715 FSC A gof ZAH Figure 3 6 Use of Zoom Tool for a close up view of the lymphocyte population in a human peripheral blood sample 2 Use the Zoom Tool to expand the view of the lymphocyte population Click on the Zoom Tool button the symbol will turn blue and use
55. Range 10 16 777 215 Apply OK Cancel Figure 3 36 Setting Min and Max channel values for the X and Y axes using the Plot Spec dialog box Open the Plot Spec dialog box for Plot 6 Under X Axis set Min Value 100 and Max Value 1 000 000 Under Y Axis set Min Value 50 and Max Value 500 000 Select the Apply button to view the changes and then the OK button to close the Plot Spec dialog box O p 10 See Figure 3 37 for results of axes range adjustments Plot 2 Plot 6 Gate R1 in P71 in ally Gate R1 in P71 in ally CD4 FE A CDS APC A COS FITC A COS FITC 4 Figure 3 37 Result of axes view adjustment to 4 logs for Plots 2 and 6 www AccuriCytometers com Page 35 Chapter 4 Using the Analyze Tab The Analyze tab is an easy way to view multiple data files simultaneously using the same plots and gating strategy In this example of human peripheral blood analysis the Analyze tab will be used to determine the percentage of CD45 CD3 CD8 and CD45 CD3 CD4 cells 1 Click on the Analyze tab near the top of the CFlow Workspace just above the 96 well sample grid 2 In the Analyze workspace plots can be copied from the Collect tab or new plots can be created Figure 4 1 For this example plots will first be copied from Collect 3 Click the Copy Plots from Collect button and a dialog box will open Figure 4 2 FP HFE 4 Color Evarnple CRloae Select to Copy Plots from the Collect
56. Tab wat Color Compensa Figure 4 1 The Analyze Tab 4 Select the check boxes corresponding to the desired plots For this example choose Plot 1 Plot 2 Plot 3 and Plot 6 Figure 4 2 Gating strategies applied in the Collect tab are not automatically applied in the Analyze tab Click OK The dialogue box will close and the selected plots will be added to the Plot List Figure 4 3 Click on the upper left plot corral in the Analyze tab to select it background turns from blue to white and then select Plot 1C FSC A SSC A from the Plot List on the left hand side of the page Figure 4 3 Note that the gate around the lymphocyte population is now named P2 Figure 4 4 P2 is a copy of P1 from the Collect tab P2 can now be adjusted in the Analyze tab without changing the position of P1 in Collect tab 6 One by one select the next 2 empty plot corrals in the top row and choose Plot 3C FL1 A FL3 A and Plot 6C FL1 A FL4 A respectively from the Plot List Figure 4 4 7 Display the data for Sample well A1 in each of these plots by clicking on the plot then clicking on well A1 Figure 4 4 On Plot 3C click on the Gate button and select to include P2 on all events On Plot 6C click on the Gate button and select to include R2 in P2 in all Figure 4 5 www AccuriCytometers com Page 36 10 In the second row of plot corrals open Plots 1C 3C and 6C this time choosing Sample
57. UN buiton a lj x Edt Ospley Incdrumert Abat Cflow sampler Auto Collect ee AT Events are being recorded Piate Name Data Capacity Used 01 02 03 OF 06 pOl 07 OS OV DIOODID Et 2 3 E4 ESI amp EF El Eo EM Ett E12 Select Plot Ma INTERRUPT PLATE ABORT WELL Figure 7 12 The Run Display while collecting data Within Auto Collect CSampler will always start collecting from position A1 If A1 is not selected as part of a set CSampler will advance either horizontally or vertically to the first well that has settings applied to it and begin acquiring data As data is acquired and the CSampler advances through the plate or tube rack completed samples will display a small blue square in the upper left corner of the well The red box indicates which sample the CSampler is currently acquiring data from The current well will also flash blue during data acquisition similar to CFlow Plus When the plate or all of the sets of samples have finished DONE will be prominently displayed across the Run Display window Figure 7 13 www AccuriCytometers com Page 66 eas Fie Edt Gaplay Iniroment About Clow Sampler accun Auto Collect Plate Typa 34 wiil piate Eject Plate A1 2 CS t t t k Re TET ampier IS MOVING tO NEXT tas Salach Ay Cibclick b view Game settings Cesnet A i 7 GT Be Bo BD a iF CV G2 63 Cb csi oe ee cal ee ee ea ei E 011 OS De ee Ge ee e
58. a MPD CEH GATED M i aij Fluidics z fie O iium C Pee Fira Ria t pie Caie Bice 10 ym Plot Seep HPO OHS GATE F167 al oo Tim noc Wam batoe Calwting DO Merits ifie N 6 Drami t Dati Capacity Wiad D Hw r iul oo Aa i 90 000 000 Evans Figure 3 16 Sample number 2 has been added to well A2 in the CFlow Workspace 4 Change the axes labels to reflect the antibody staining or fluorochrome used Clicking on any axis label will cause a menu to appear Select the last option Rename Parameters Figure 3 17 ACCU Fia i fienele HFE CEAS HATES F1 in wi z Aa i Sees HPO CEA BATE t irs al Piet p Tampi Mr CL GATE 1 i ail x frata Ti p Rename Paranerters a PL Cy PEJ Yireare z Fiii FLA bo PLAN firea apphy bo G Samo KPBCOISPECY7 ox Cancel 2 All cepi all ae 7 eS g Find ki Somple HPE Cihat GATE MH in wl 3 i 1 Click on axis label and select Rename Enter axis label into the appropriate text Parameters from the drop down menu box and click OK Figure 3 17 Changing axes labels using the axis label drop down menu and the Rename Parameters option 5 Enter the label next to the parameter to be renamed in the Rename Parameters dialog box In this example CD45 PE Cy7 is entered The label can be applied to the current sample or to all samples by choosing the appropriate radio button at the b
59. ab settings such as Run Limits Thresholds and Color Compensation can be applied to either the current sample being viewed indicated by a red box surrounding the well or to all the samples in the sample grid 2 To assist in setting up experiments that will be processed using the Auto Collect Tab The following can be done using the Manual Collect Tab prior to swapping to the Auto Collect Tab a Plot creation b Gating schemes c Setting threshold values primary threshold channel trigger secondary threshold channel d Color compensation e Run Limit settings applied to individual wells from the Manual Collect tab are not carried over into the Auto Collect tab 3 The Manual Collect tab can also be used to collect data from a plate if the plate has been interrupted or aborted in the Auto Collect Tab All of the plot tools available in CFlow Plus are available in the Manual Collect tab Creating plots regions gating schemes and other analysis features are described in Chapter 3 www AccuriCytometers com Page 60 The Auto Collect Tab The Auto Collect tab Figure 7 7 is designed to facilitate the automatic running of multiple samples from a well plate or the sample tube rack There are two fundamental differences between the Manual Collect tab and the Auto Collect tab The first is that the Auto Collect tab does not allow the creation of plots This has been replaced with the ability to easily label a large number of samples Th
60. agesss 59 Figure 7 5 The Run Unclog Cycle Window cccececccccececeseeseeeeeeeeeeeeeeeceeeeeesseeeeeeeeeeesseeaseeeeeeeeessaaageeeeees 59 Figure 7 6 The Calibrate Fluidics WINKOW cccccsseeeeccceceeceaeeeseeceeeceaaeeseceeeeessuaeeeueeeeeeessaaeeeeeeeeesssaaaasses 60 Figure 7 7 The Auto Collect tab cccccccccccccccceeceeeeeeeeeeeeeeeeeeeseeeeeeeesaeeeseeeeeseseeeaseeeeeeeessseeaseeeseeeeesssaaageses 61 Figure 7 8 Selecting an individual sample cccccceeeececccceeeeeeeseeeeeeeesseeeeseceeeeeeeaaaeeeeeeeeeessaaesseeeseeeessaaageses 62 Figure 7 9 Applying settings to a set of samples will turn those sample wells the same color 63 Figure 7 10 The Auto Collect tab displaying two Sets cccccscccceeecssesseeeceeeessseeeeseeeeeeeeseeeesseeeeeeeessaeaneses 63 Figure 7 11 The Run Display cccccccccccseeeeeeseeeeeeeeeeeeeseeeeeeseaeeeeseaeessaeeeeessaseeessaueeessauseeessaseeeessaneessaaesees 65 Figure 7 12 The Run Display while Collecting Aata ccccccseseccccsseeeeseeeecseeeeeeeeeeeeeesaeeeeesaaeeessaneeeessaeees 66 Figure 7 13 The CSampler display after completion of a sample FUN cccceceeseeeeeeeeeeeeeeeeeeeeeeeeesaaeeeees 67 Figure 7 14 Collision detected WINdOW cccccccsssssseecceeeeecaeeeeeeceeeeeseeeeeeeeeeeessuaaeseeeeeeeessaaaseeeeeeeeessaaaaeses 69 www AccuriCytometers com Page 3 Chapter 1 The Basics Operating the Accuri C6 Flow Cytometer
61. ans Ctril R HPB CD45 PE Cy 123 4 5 6 8 9 10 11 12 HEE A4 A5 AB A7 AB AO A10 A11 A12 Plot 1 Sample HPB CD45 Plot 2 Sample HPB CD45 Plot 3 Sample HPB CD45 GATE No Gating GATE Pin all GATE P1 in all B1 B2 B3 B4 BS BG B7 BS BY B10 B11 B12 C1 C2 C3 gt C4 C5 cep c C8 C9 C10 C11 C12 D1 D2 D3 D4 DS DE DF DS DY P10 D11 D12 E1 E2 E3 E4 ES E6 E7 E8 f E9 E10 E11 E12 lt 6 wi ao i a D F4 F2 FS F4 FS FO FF FS FO FIO F11 F12 G1 G2 G3 64 65 GB G7 G8 69 610 611 G12 200 000 465 000 H1 H2 H3 H4 HS H6 H7 HS H9 H10 H11 H12 FSC A 10 C6 and CFlow are connected ram O SPEC OQ H eo and ready po D TNn oM oe p E Plot 4 Sample HPB CD45 Vorm Plot 5 Sample HPB CD45 Vil Plot 6 Sample HPB CD45 Y Figure 3 24 Quadrant Q2 is selected with mouse click indicated by bold red and Calculate Median chosen from the Display drop down menu 5 The median fluorescence channel value for the events in each quadrant will now appear in the Statistics Table Figure 3 25 However no median is calculated for the top line named This Plot CFlow only calculates median values for events encompassed by Regions Markers Quadrants or Polygons www AccuriCytometers com Page 28 I Plot 3 Sample A Median FL1 A Median CD45 PE CyT A A Gated on F1 in all This ra 101 660 7a ean es e ee 771 0 TL ote ah 10 2000 1 322 0 Figure 3 25 Statistics Table up
62. area and a dialog box will open Set the Primary Threshold on FSC H to 80 000 and Apply to All Samples This threshold should work well to gate out debris and noise from cell samples freshly isolated from human or mouse tissues A lower FSC H threshold may be needed to effectively analyze small cells such as platelets or bacteria A higher FSC H threshold may be needed when working with large cells such as cell lines The threshold settings can be raised or lowered at any time during analysis If raising a threshold setting will result in data loss CFlow will give a warning Figure 3 4 10 11 12 E Run Limits Fluidics PRIMARY THRESHOLD i Run Unlimited Slow w Permanently eliminate events on Flow Rate FSC H 7 less than 80000 Range Minimum 10 IV 50000 events Core Size amp Custom h ungated Sample gt OPTIONAL SECONDARY THRESHOLD Permanently eliminate additional events on m f iir f eee gd ale less than 0 Range Minimum 10 fo O pL Apply to Only this sample Core Size Apply ose n All samples Backflush Unclog Threshold 407 0 a m Set Threshold CD4 PE A 80 000 on FSC H BF None aa C _ SPEC A ast Riin Cumulative Malata Camnala Matal Figure 3 3 Threshold Settings dialog box 62163 64 65 66 67 68 6a le106111612 H0 H2 H3 H4 HS H6 H xX F These settings will Threshold Seu PERMANENTLY DELETE eo 8113 events from this sa
63. ay not encompass the main population of bead events on the FSC H vs SSC H plot This will be addressed in Analyzing and Recording Your Validation Bead Data 17 Name this sample including the date processed in the text box just above the Sample Grid Samples can be named at any time before during or after collection 18 Remove the beads tube and wipe off the end of the SIP with a Kimwipe or comparable material to minimize bead carryover To further minimize carryover click Backflush while holding a reservoir or empty tube under the SIP Fluid should drip or stream out of the SIP 19 Place a tube with suspended 6 peak beads on the SIP Vortex the tube prior to use 20 Click on well E1 to advance to well E1 Verify that the Events check box is still selected and set to 50 000 events in Ungated Sample 21 Click RUN The R2 region on the FSC H vs SSC H plot in the second row of the CFlow bead file may not encompass the main population of beads 22 Name this sample including the current date in the text box just above the Sample Grid 23 Remove the beads tube from the SIP and wipe the end of the SIP 24 Place a tube with 2 mL of filtered deionized H2O on the SIP and advance to any empty data well 25 Select the time check box Min Sec in the Instrument Control Panel and set it for 2 minutes Click RUN 26 Leave the tube on the SIP after the C6 stops running Always end each data collection session on the C6 by running filtere
64. ct data from Sample 2 the CD45 PE Cy7 tube Sample 1 is removed from the SIP and replaced with Sample 2 after gentle re suspension There is no need to Backflush between samples Residual sample in the SIP and flow cell is cleaned out automatically before the C6 begins data collection You may wish to wipe the outside of the SIP with a lab wipe to minimize carryover between samples 2 Click on an empty data well in the 96 well sample grid well A2 is used in this example The newly selected well is now outlined in red The plots and gates created previously will remain but will not contain data www AccuriCytometers com Page 24 3 Click the RUN button to start sample collection During data collection data is displayed and actively updated in the plots When the run to limit is reached the C6 stops sampling from the tube and well A2 turns blue to indicate that it now contains data Figure 3 16 SS Wen amo CDMS Ad A2 criowa File Edit Gipplay instrument A Fist Sampii HPO Cote ATE M n all Mara Sarge HPD COA s 12 3 4 3 T A Wa ie 2a ag a oe ac a 10 11 13 AE Bi Bz i BS BY BS Be BY Bal Be Bo Bel Bir ce be Ag abo a tie B C2 ta C4 08 Of cr Co Oho Ctl CEZ i bap Ca faj ps i oe o7 PE ee Ded MEE E E1 E EX E1 ES E0 E7 Be E Bt Ett Bee F ee ee ki ea Se E E ee a a a i a H Hi Ht HI HAI HS HE H7 HE Ho HES HIT HEX Met amp ping
65. d deionized H2O for 2 minutes Leave this tube on the SIP between sessions and when shutting off the C6 Saving the Data Saving data as a CFlow file will save the CFlow workspace in its entirety including collected sample data plot layouts gating color compensation and threshold settings that have been added Changes made in the Collect Analysis or Statistics tabs will be saved in a single comprehensive and often large data file Data collected on the C6 should always be saved as a CFlow file There is no need to export after every sample run Data from individual sample wells can be exported from CFlow as FCS 3 0 files at any time www AccuriCytometers com Page 7 Save Your Data as a CFlow File 1 In CFlow under the File menu select Save CFlow File Figure 1 3 and save the bead data file as a Validation Bead File Use this file each day to collect Validation Bead data advancing to a new well in row A or E for 8 peak and 6 peak beads respectively Include the date in the Sample Naming Field when naming each day s bead sample CAUTION CFlow does not automatically save CFlow files to the computer hard drive The data is saved locally in RAM until it is saved as a CFlow file or exported as FCS files CFlow allows switching between data wells to view data even when data is not stored to the hard drive 2 End every session of data collection on the C6 by placing a tube containing 2 mL of filtered deionized H2O on the SIP
66. d logarithmically while light scatter parameters are displayed linearly Figure 3 9 a b Click on the Plot Spec Tool select the appropriate parameter from the X axis and Y axis menus and change the radio button selection from linear to log display Figure 3 9 b www AccuriCytometers com Page 21 Click on Plot Plot 1 Sample Unstained Plot 2 Sample Unstained Plot Spec dialog Spec Tool GATE No Gating Hs E i box S Click on axis i label H Set Plot Specs for Plot 2 T pE Plot 2 Sample Uhstained No Gai AXIS GATE No Gating FLi V linear Min Yalue Max Value log Hide 1st 10 16 777 215 Iv He Range 10 16 777 215 Y AXIS FL2 A Y linear Min Yalue Max Value log Hide 1st 10 16 777 215 lv PREHR Range 10 16 777 215 Apply OK Cancel Q 7 i 16 7772 200 000 l 000 000 10 000 000 16 777 215 on FSC A FSC 4 o Ct S SSC A FL2 A FL3 A FL4 A SSC A 000 000 10 000 000 0 Height Width Time 0 5 000 000 10 000 0p0 16 777 215 FSC A Rename Parameters a Parameter axis menu b Plot Spec dialog box Figure 3 9 Changing density plot axes parameters and scaling Gate button Gate dialog box Change Gating for Plot 2 tak Gating choices Include _ Exclude E Intersection Show Vertical Markers Show Quadrant Markers Plot 1 Sample Unstained GATE No Gating P1 FSC A SSC A P1 onall
67. d to the entire plate or tube rack and can not be applied to individual wells Run Direction Data can be collected from plates in either the horizontal A1 A2 A3 etc direction or the vertical A1 B1 C1 etc direction This is determined by choosing the Run Horizontally or Run Vertically toggle beneath the Settings Controls in the Control Panel www AccuriCytometers com Page 64 The Run Display The Run Display is used during data collection in the Auto Collect tab To access the Run Display window click the OPEN RUN DISPLAY buiton This button is only active after one or more wells have had settings applied Once the Run Display is opened the Sample Notation Table will be replaced with the Run Display The Run Display contains a single larger plot the Sample View Plot an additional traffic light status indicator the data acquisition counters and several Pause alternatives Figure 7 11 The Control Panel is disabled while the Run Display is open In order to return access to the Control Panel the Run Display must be closed using the CLOSE RUN DISPLAY button lit x de ACCU Status Indicator Sample View Plot Sample Plot Menu Interrupt and Abort Close Run Display Figure 7 11 The Run Display Sample View Plot All plots created in the Manual Collect tab are listed in the Select Plot drop down menu beneath the plot window and can be selected at any time during data collection One plot at a time ca
68. dated with median values after choosing Calculate Median from the Display drop down menu Color Compensation values to correct for fluorescence spillover will be calculated in this example using the median comparison method outlined in the steps below An alternative method is to utilize the C Comp Calculator Excel Spreadsheet provided by Accuri Use of the C Comp Calculator saves time and improves accuracy when determining fluorescence subtraction values 6 Click on the Set Color Compensation button to open the Compensation Settings dialog box Figure 3 26 lower left Plot 1 Plot 3 Sample 42 2 GATI 2 Compensation A a GATED P1 in all X had i m amponnnrar A Settings Correct FL1 by subtracting S 33 B4 BS B B7 BS B9 B10 B11 B12 FL3 33 f c4 f cs f ce f c7 f cs f co c10 C11 C12 o q a ESA gt 23 D4 DS DB DF DS D9 D10 D11 D12 lt 3 Ri a wo gt a 3 E4 E5 E6 EF E8 E9 E10 E11 E12 x gS oe a 3 F4 FS F6 FF FS FO FIO F11 F12 oe _ i oq 33 64 65 GB G67 68 69 610 611 612 o Compensation Settings for Sample A2 x 3 43 H4 HS HE H7 H8 HO H10 H11 H12 Correct FL1 by subtracting a percentage of 250 FL2 P o Yo FL3 p o0 Yo FL4 0 00 CE Cytometer not connected Correct FL2 by subtracting a percentage of E E Plot 6 Sample 42 pe Fluidics a FEL 0 00 FL3 0 00 FL4 fo 00 hy g GATE P1 in all Siow Medium Fast z eT one X f ONR Sten amin e orrec y subtracting a percentage o 2 in
69. e ee a ee Ep tt G2 0a b OS Oe ee ee CoCr caer ra a ae rS Pe Oe Pe aaa a E GPO l oil oal oa oa f OP da aa lomila ia l Hii Hapag ee ee a r l a l a Delati parsi on Miner ty Faen 7 lati than fosa bid bee thar Figure 7 13 The CSampler display after completion of a sample run Once a plate is complete the Run Display must be closed in order to continue using CFlow Sampler The Run Display is closed by clicking the CLOSE RUN DISPLAY button This will return the Sample Annotation Table to the screen Analysis can then be completed using the Analysis and Statistics tabs Analyze and Statistics Tabs The Analyze and Statistics tabs are used in the same manner as CFlow Plus Please see Chapter 3 for a complete description of these tabs www AccuriCytometers com Page 67 Running a Sample Plate Example The following is intended to provide an example of a typical setup and data collection workflow using CFlowSampler software 1 Open CFlow Sampler software 2 Select the Manual Collect tab 3 Select a plate type from the Plate Type Menu 4 Create the plots that are going to be needed for running the plate or load the template 5 If necessary acquire some events to define regions gating etc 6 Switch to the Auto Collect tab 7 Select the wells in the Sample Grid where data will be collected 8 Set the desired acquisition settings such as number of events threshold washes needed etc 9 A
70. e Tab view after scrolling down to empty plot Corrals ccccccseeeeeeeeeeeeeeeeeeeneeeeeeeeas 39 Figure 4 8 Analyze Tab view after adding 2 more rows of Plots 1C 3C and 6C and populating with data Hom Sample A3 and AA eect ta to acecalee aE NEN T a d AER 40 Figure 4 9 Analyze Tab Plots are selected for printing ccccccsesscccccceseeeeeeseeeeceeeeaeeeeeeeesseeeeeeesseeeeeeeeaas 40 Figure 4 10 Analyze tab Overlay Tool has been selected cccccccceseeseeeeceeeeeeeeeeeeeeeeessaeeeeeeeeesaeeeeeeeeas 41 Figure 4 11 Analyze tab Overlays of FL1 A data for samples A2 A3 and A4 The Overlay Legend has DESTO ENE lenei E EE E E AE EAE ee eee E A ee eee 41 Figure 5 1 The File MenU s snnonnnnsennnnsrnnnssrnensrnresnrrensrnresrrersrnresnrrensrnrosarrrrsnnresarrersnnrernrrersnrreenrrererrrerne 42 Fig r 5 2 The Edit Ment oxi retirees tanto saeen et ane ciceatatedties EEE A AA Ea TAEAE 43 Figure 5 3 Rename Parameters wicca cteinetasstncutancsteneharisntetnadtisiataieeband susuannddemctaussanetaedsteataksdnotubantencaaueinceldads 43 Figure 5 4 Rename Parameters selection options MENU cccssesecccseeeeeeeeeeeeeeeeeeeseeeeeeeeaueeeeseeeeeesaeneeeas 44 Figure 5 5 The Display M nu cccccssccccsssececcesseccseeseeecaseeeceauseecssasececsaeeeeseuseeeseageeessageeesseuseseesaseeessageees 44 Figure 5 6 Events Display Settings ccccccccccccecsseeseeeeeeeeeeeeesseeeeeeeeeeeeeseeeeeeeesseeeaeseeeeessseeaeee
71. e second difference is the ability to create sets of samples HJ Untitled Workspace CFlow a laj a x Fie Edit Display Instrument About CfFlow Sampler Auto Collect Plate Type EIRtENaric Eject Plate Al Plate Name i 42 Select All A3 Ctrl click to view sample settings Deselect All Well Sample Name Rename FL3 Rename FL4 Notes Rename FL1 Rename FL2 At Al a1 az asl aal asl as a7 as aa arolas a12 AS 46 a 43 49 410 All 412 B1 B2 B3 B1 B2 B3 B4 BS B BF BS BI B10 B11 B12 C1 c2 gt c3 gt C4 cs C6 Cc C8 C9 010 011 C12 D1 D2 D3 D4 DS DB D7 DB DY D10 011 D12 E1 E2 E3 E4 ES EG E7 E8 E9 E10 E11 E12 Fi F28 F38 F49 FS FO FF FS FO F10 F114 F12 61 62 G3 G4 G5 GB G7 G8 G9 610 611 G12 H1 H2 H3 H4 HS HG H HS H9 H10 H11 H12 C6 and CFlow are connected and ready Run Limits Fluidics B4 P Run Unlimited Slow Medium Fast BS fioooo Flow Rate 14 pLimin M 10000 Fis Core Size 10 pm B in Ungatea Sample Y Set Threshold B7 E fo Min fo Sec Delete events on Minimum 10 BS O bo a FSc H x less than e0000 B9 Wash Settings x tessthan fo B10 None p Bil 1 cycle takes about 1 min B12 ei 35 Remove Settings G2 Agitate Plate Run Horizontally c3 None even I min C Run Vertically et 1 cycle takes about 0 5 sec c5 c EF c8 cg C10 cii ciz D1 D2 Figure 7 7 T
72. ed to show channels 0 to 1 600 000 on FSC A and 0 to 800 000 on SSC A Click on the Plot Spec Tool lower right hand corner of Plot to change the axes Channel range or to view axes on a logarithmic scale Figure 3 2 I tf FF 4 5 amp 7 g F 10 11 17 Me a a Tb ee re 2 2 04 05 OF Cr Oo te cofit BD tai a ee a ei ea AT Ei G e E Wapi Ef GF EA EG E9 E EB Ee Een Ete Po PO Pee PS PB pral ro ranr gil a ek gal os aa ae oe og ona eae H HII Hd ee PH A H f ee OH 1 Te Ci mo Cea are oer ed aca mady penne Set FSC H Threshold to 80 000 ists Dale Sampii Daia T non Tias om P Wam pea deleting Of Menines j Com Capectr load O60 Cyente fyb frat O af 10 DADA Laser Figure 3 1 CFlow Workspace after selecting File New CFlow File from the CFlow file menu T Plot 1 Sample HPB CD3 Set Plot Specs for Plot 1 x GATE No Gating SORE Choose Linear or log scale Fsc aW linear Min Value Max Value log o 1 600 000 pene a Range 0 16 97 Y AXIS SSC A 200 000 400 000 600 000 800 000 EZI Saal Set axes channel ranges Min Value Max Value log 0 oo 800 000 Hide 1st 0 800 000 IV esata Range 0 16 777 215 la 5 om oN H Apply OK Cancel D 500 000 1 000 000 1 600 000 FSC A Figure 3 2 The Plot Spec Dialog Box Changing these settings only affects the view of the data No data is changed in
73. eded to collect data Before data collection can begin the Traffic Light below the Sample grid must be green and the words C6 Cytometer is connected and ready appear in the message box Getting Started This section provides a step by step guide for running validation beads The validation will demonstrate many of CFlow features and ensure the C6 is working properly before running experimental samples All the individual features of CFlow are described in detail in Chapter 4 Complete the initial instrument set up as outlined in the C6 Flow Cytometer Instrument Manual Section 4 before continuing Starting CFlow Open CFlow by double clicking on the CFlow icon on the desktop The message box in CFlow will say Cytometer Not Connected C6 Startup Procedure 1 Check sheath bottle blue ring fluid level and fill if needed Accuri recommends the use of 0 2 um filtered deionized H2O with bacteriostat added Accuri Part KR 220 CFlow will alert the user if the bottles need attention when the C6 is turned on and CFlow is running Empty the waste tank red ring Check Cleaner green ring and Decontamination Solution yellow ring bottles Fill if necessary www AccuriCytometers com Page 4 Refer to C6 Flow Cytometer Instrument Manual Section 4 3 F CtFlow Plus 96 Well Sample Grid Status Bar Traffic Light and Message Box Custom Fluidics Settings User Defined Flow Rate and Core Size User Defined Stop Criteria
74. eeeeseeeeesseceeeeeseaaeeeeeeeeeeessaaeeseeeeess 16 Chapter 3 Using the Collect Tab cccccccccccccsssssseecceeeeeeeeeeeceeeeeseeeeseceeeeeesssaeseeseeeessueeaseceeeeessssaaaseeeeeeees 17 MOGUC ION sess cratteti cece cons soese EE te sec satire E E E E E T seaceesteeseneweseoseue 17 Experimental Design of Tutorial File ccccccccecccesssseeeeeeeeeesseeeeeseeeeeeseeeeeeeeeseeeseaeeessaeeeeeseeseeensneesens 17 Opening a New CFlow Workspace and Collecting a Data File cccceecseeceeeeeeeeeeeseeeeeeeeeaeeseeeeeees 18 Creating Plots and Applying GateS cccccccccccccssssseeeeeeeeeseeeesseeeeeeeeeeeeseeceeeeseseeeseeeeeeeeeseaseeeeeeeeeesseaaaaesss 20 Adding Data to a CFlow File and Naming Plot AX S 0 ccc ceeeeeeeeeee eee eeeeeaeeeeeeeeeeeeeeeeeeeeeeaaaaaaaaeeeeeeees 24 Naming and Saving a CFIOW File cccceecccccceeeeeeeeeseeceeeeeeeaaeeeeeeeeeeseeeasseeeeeeeessueeseeeeeesseeasseseeseeeeeeaaas 26 Calculating Median Values and Applying Fluorescence Compensation cccssssscceecseeeeeeeeeaeeeeeeeeas 27 Adding and Applying Additional Gates ccccccccccccecccssseeeceeeeeeseeeeseeeeeeeesseeeeeeeseesseeeaseeeeeeeessaaeaeeseeeees 32 Adjusting the Axes View Using the Plot Spec Tool ZOOMING ccccceecesseeeeeeeeeeeeeeeeeeeeeeesesaaaeeeeeess 34 Chapter 4 Using the Analyze Tab ccccccccccccccsssseeecceeeeceaeeeeeeeeeseseaseueeeeeeessaeasseseeeeeesseaseeceeeeesessuaaaeeeeeeess 36 Make an Overlay
75. eipt Refer to Reagents Required for ordering information Validate the performance of the C6 with validation beads each day that the cytometer is used The C6 should be able to distinguish a defined number of peaks within each fluorescence channel If these peaks are not discernable there may be a problem and any data collected may be flawed It is important to collect and save a bead data file every day that data is collected The user can easily address many C6 issues with assistance from Accuri Technical Support If a problem is experienced that requires telephone assistance Technical Support may ask for data from the bead file to assist in troubleshooting Reagents Required 1 Spherotech 8 Peak Validation Beads Accuri Part QA 100 or the prediluted 12x75 mm tube from Accuri labeled 8 Peak Beads 2 Spherotech 6 Peak Validation Beads Accuri Part QA 110 or the prediluted 12x75 mm tube from Accuri labeled 6 Peak Beads 3 Sheath fluid Deionized filtered water 0 2 um filter plus Bacteriostatic Concentration Solution Accuri Part KR 220 Running the Bead Samples 1 Be sure that the C6 has been switched on for at least 15 minutes 2 Verify that the file named Bead Template c6t has been copied to the computer attached to the C6 This file is on the CFlow CD shipped with the C6 and also available on the Accuri website Double click on the bead template file to open or load the file from the CFlow File Menu Place an empty 12x
76. eviously exported from another CFlow file to the currently open workspace Only FCS files that were created using a C6 can be imported into CFlow Export FCS File Exports and saves the currently selected data well sample as a FCS 3 0 file to a user selected folder Export ALL Samples as FCS Saves all the data wells samples as individual FCS 3 0 files in the folder CFlow FCS Exports on the desktop Print Selected Items Prints the selected plots and associated statistics At least one plot should be checked in the Print check box located in the upper left hand corner of each plot for this option to be enabled The Statistics Box associated with each selected plot will automatically be selected for printing The selected plots will print Landscape orientation on the first page with the associated statistics in Landscape orientation on the second page The Print check box can be unchecked for any given Statistics Box in order to print plots alone Quit Quits CFlow and closes the application CFlow will always prompt to Save the Workspace upon selecting this option www AccuriCytometers com Page 42 The Edit Menu FA untitled Workspace CFlowm File Edit Display Instrument About Undo Ctrlt z Redo Chrl Copy Chrl c Paste Ctrl Rename Parameters 1 2 3 4 5 6 7 B 9 10 11 12 a AS AS Ad AS AG AFT AS AD AIO A11 ATZ Bi Bb BS B4 B5 BE BF B BS B10 B11 Biz Ci C2 gt Cs C4 CS Ce Cr Ce CS C10 C11
77. for a sample volume of 400 uL in a standard 12x75 mm tube during manufacture Accuri suggests that you recalibrate your system for your most common tube type and volume to increase the accuracy of volume measurements If the C6 is being used routinely for volume analysis www AccuriCytometers com Page 14 perform volume calibration once a week For intermittent volume analysis perform volume calibration prior to each set of experiments requiring it Volume measurements can only be performed in CFlow Plus software To ensure the most accurate volume measurements e The peristaltic pump tubing must be less than two months old e Run samples at Medium or Fast speeds e Calibrate the C6 to an equivalent sample volume fluid height in tube as the sample to be measured e Never perform calibration with less than 150uL in any type of sample tube To Calibrate the C6 1 Place a tube of the same kind to be used in the experiment and containing a volume of sheath fluid close to the sample volume in the experiment 65 uL on the SIP For example if cell samples are diluted to 400 uL in 12x75 mm tubes then the Fluidics Calibration should be performed with a 12x75 mm tube containing 465 uL of sheath The minimum sample volume to calibrate a 12x75 mm tube is 150 uL run calibrate with 215 uL in the sample tube 2 Under the Instrument Menu choose Calibrate fluidics The fluidics calibration will take 5 minutes If calibration fails the C6 will re
78. from the Display drop down MenU eee ee eee eee nen ee ne ee ee 29 Figure 3 26 Opening and using the Compensation Settings dialog DOK cccseeeeeeeeeeeseeeseeeeeeeeeeeaeeeeees 29 Figure 3 27 Results of subtracting spillover of FL8 PE Cy7 signal from FL1 by 1 35 Note that the median FL1 channel numbers in Q1 UL 238 and Q1 LL 231 are approximately equal blue boxes 29 Figure 3 28 Results of selecting Plot 4 applying Quadrant markers and calculating Median fluorescence channel valles oseaan eneen NEEE ENAA EE E ANEN EEEN na EE 30 Figure 3 29 Results of correcting FL2 by subtracting 4 3 of the signal from FL3 PE Cy7 The Median FL2 fluorescence channel value for Q2 UL 131 is approximately equal to the Median FL2 fluorescence channel value for Q2 LL 134 cciecsescescncsnicadeccinsasravatdeoseesexctinntwseee sacdudueddaserdistalrsocctdavergaceepandandzccedennsbeadoesuaeesee 31 Figure 3 30 No fluorescence subtraction of FL3 PE Cy7 signal is needed from FL4 as indicated by comparison of Median FL4 values for Q3 UL 290 and Q3 LL 263 2 0 eeeccccceseeeeeeeeeeeeeseseeeeeeeeeaes 31 Figure 3 31 Correction of spillover from FITC FL1 into FL2 a Before fluorescence subtraction b After calculation and application of appropriate percentage subtraction of FL1 from FL2 sssaesesennenennnnnennnnne 32 Figure 3 32 Gating region R1 has been drawn on Plot 3 to include the CD45 PE Cy7 positive events 32 Figure 3 33 Gating Plot 2 C
79. gn plot www AccuriCytometers com Page 53 17 The sample with VirtualGain applied will now have a black asterisk under the parameter label The negative peak for Sample 2 now has a MFI 26 7 Without VirtualGain the MFI was 73 4 Figure 5 9 Plot 11 Sample 43 GATE Pe Figure 6 9 The sample with VirtualGain applied has a black asterisk under the parameter label 18 To switch between the VirtualGain and native displays click the asterisk A black asterisk indicates VirtualGain applied a grey asterisk indicates display of the original data Figure 5 10 Plot 11 Sample 43 Plot 11 Sample 43 GATE Pe GATE Pe Figure 6 10 Click the asterisk to switch between VirtualGain and native displays A grey asterisk indicated native data a black one Virtual Gain applied 19 Overlays will automatically display VirtualGain when applied Sample 2 now lines up with the Control and Sample 1 The VirtualGain asterisk is not displayed in Overlays when VirtualGain is applied to some or all of the samples in the overlay Figure 5 11 www AccuriCytometers com Page 54 Flot 12 Sample 41 05 42 GATE Fe Figure 6 11 Overlays automatically display VirtualGain whenever applied 20 To remove VirtualGain select Remove All VirtualGain from the Display menu This will remove VirtualGain from every parameter in every sample within the CFlow file www AccuriCytometers com Page 55 Chapter 7 CFlow Sampler Introduction
80. he Auto Collect tab Sample Annotation Table The right side of the Auto Collect tab contains the Sample Annotation Table This table allows for the naming of samples renaming of parameters and adding a specific notation to each sample Information can be entered manually for each individual sample or directly imported from a spreadsheet program using the Copy Ctrl C and Paste Ctrl V commands The table layout is fixed and therefore sample names must be entered across rows 1 12 before moving down the columns A H of the sample grid eke Individual fields can be selected by clicking in them with the mouse or advancing with the Tab key The keyboard arrow keys do not function in this view Text in a field can be select by double clicking in the field When the Open Run Display button is selected the Sample Annotation Table is replaced with a new www AccuriCytometers com Page 61 window containing a single plot This plot is described in the Run Display section Creating Sample Sets and Applying Acquisition Settings Selecting Samples A sample well can be selected by clicking on it with the mouse Unlike the Manual Collect tab where selected wells are indicated by a red box selected wells in the Auto Collect tab will contain a black checkmark tick as seen in Figure 7 8 Creating a Set of Wells Samples A set is a grouping of samples that will be acquired using the same acquisition criteria number of events
81. he SIP In CFlow Deselect the Run Unlimited check box and select the Time check box Enter 2 minutes Click RUN Wait for run to finish Click DELETE EVENTS FROM SAMPLE 0 Keep the tube with water on the SIP until experimental samples are ready to be analyzed oe et ee ot Care After Running Samples Always perform this process after the last sample to ensure cells or other particles are not left in the SIP Place a tube with 2 mL of Cleaning Solution on the SIP Select an empty data well Select the Time check box Enter 2 minutes Click RUN Wait for run to finish Remove the tube of Cleaning Solution and place a tube of 2 mL filtered deionized H2O on the SIP select the Time check box and enter 2 minutes Click RUN When run is finished leave the tube of water on the SIP O N ee SS NS Instrument Shut Down 1 Perform Care After Running Samples procedure detailed above 2 Press the Power Button on the front of the C6 3 When the Power Off process is completed the CFlow Message Box will read C6 Cytometer not connected Validation of Instrument Performance Follow the directions in Chapter 1 for running the 8 and 6 peak SPHERO Calibration Particles Use the Accuri C6 Cytometer Log spreadsheet located on the CFlow Installation CD to record top peak CVs and Mean channel numbers for each fluorescence detector Calibrating the Fluidics System for Precise Volume Measurements The C6 fluidics system is calibrated
82. he dialog box shown in Figure 4 11 All plots created in the Analyze and Collect tabs including Overlays can be dragged and dropped into most Microsoft Office compatible programs Statistics Tables can be copied and pasted as well www AccuriCytometers com Page 41 Chapter 5 CFlow Software Menus The File Menu 2 Untitled Workspace CFlow File Edit Display Instrument About Open CFlow File or Template New CFlow File Save CFlow File Save as Template Import FCS File f 8 9 10 11 12 AF AB AG AIO A11 A12 Export Fics File Export ALL Samples as FCS Print Selected Items Iw B7 BS B9 B10 B11 B12 i CF Ce CS C10 C11 C12 uit DY De DS BAO D11 Baz E1 E E3 ES E5 ECR E7 E8 E9 E10 E11 E Fi g FaFa Ft FS Fe FF FS FO FIO Fld Faz G1 G2 63 64 65 Gh GF GB G8 G10 611 G12 E F G H H1 He H2 H4 HS H H7 HS HS H10 H11 At Figure 5 1 The File Menu Open CFlow File or Template Opens a pre existing CFlow file or CFlow Template Only one CFlow workspace can be opened at a time New CFlow File Opens a new blank CFlow workspace replacing the existing open workspace Save CFlow File Allows re naming and saving of the currently open CFlow workspace Save as Template Creates a template from the currently open CFlow workspace All markers regions gating parameter names sample names etc will be saved without any data points Import FCS File Allows importation of a FCS file pr
83. hreshold settings can be applied to either the sample currently being viewed or all the samples Selecting All Samples will apply these settings to all of the previously collected data in the open CFlow workspace This command is also available directly from the Control Panel via the Threshold button Threshold Settings for Sample A1 E xj PRIMARY THRESHOLD Permanently eliminate events on E less than 80000 Range Minimum 10 OPTIONAL SECONDARY THRESHOLD Permanently eliminate additional events on less than J Range Minimum 10 Apply to Only this sample FM rae Apply Close Figure 5 11 Threshold Settings Take care when setting the Primary and Secondary Thresholds When the thresholds are set before or during data collection any events not meeting the threshold criteria will be not be acquired or saved When changes are made to the threshold values after data collection a warning message will appear if changing the threshold value will result in permanent loss of data not meeting the new threshold criteria from the data file Figure 5 12 www AccuriCytometers com Page 46 62 63 cal cs l ce l 67 68 69 Ie10 611 612 of H2 HS Ha HS He H Ai nn xk These settings will SEES PERMANENTLY DELETE eee 8113 events from this sample E lits Permanently el Unlim F5c H z less These settings will also be applied as you collect additional samples Cancel v less Mir Apply to Only
84. it to encompass the area on which to zoom Figure 3 6 3 Choose the Polygonal gating tool and draw a region P1 Figure 3 7 Left click on the mouse to anchor each vertice double click to close the polygon The percentage of cells within P1 for that plot appears on the screen automatically There are two other options available for drawing gating regions the Rectilinear and Quadrant gating tools These tools will be used in other steps of this tutorial www AccuriCytometers com Page 20 Plot 1 Sample Unstained GATE No Gating Polygonal Rectilinear Quadrant Figure 3 7 Polygonal gating tool was used to draw P1 on the 2D plot above Other available gating tools are Rectilinear and Quadrant 3 Open anew plot to display the data collected for FL1 and FL2 This data is currently stored in RAM Select the Density plot icon from one of the empty plot corrals and a default FSC A vs SSC A plot appears Figure 3 8 There are two other plot options available allowing the creation of a Histogram or a Dot plot Histogram Dot Density Default FSC A vs SSC A Plot Figure 3 8 Opening a Density plot defaults to a linear FSC A vs SSC A plot 4 Change the newly opened linear FSC A vs SSC A plot to a logarithmic FL1 A vs FL2 A plot There are 2 ways to accomplish this a Click on the Axis label of the plot A menu will appear allowing the selection of the desired parameter to display By default fluorescence parameters are displaye
85. l i Dh Dh S i E fo Min 0 Sec Flow Rate a Ed g g 2 i fo o uL a a Core Size 5 ym os Sey yi Backflush Unclog Threshold T m 4 Set Threshold None 80 000 on FSC H Plot 2 Sample HPB CD3 F CD4 CD45 CD8 Count Volume pL of This Plot of All MeanCD3FITC A MeanCD4PE A CVCD3FITC A CVE pi Gated on R1 in P1 in all This Plot 99 183 0 0 100 0 43 3 23 875 3 1 762 1 62 0 Set Color Compensation Q3 UL 147 0 0 0 1 0 1 960 4 1 607 0 53 3 ee lOs IIs Q3 UR 59 194 0 0 59 7 25 8 32 131 1 2 933 7 28 5 Last Run Cumulative Delete Sample Data Q3 LL 21 455 0 0 21 6 9 4 547 0 21 5 47 8 I O Event 229 303 Y a z ae ao eee baie ala Q3 LR 18 387 0 0 18 5 8 0 24 701 0 22 5 29 2 0 0 Microliters 0 0 0 Events Sec o Data Capacity Used Plot 3 Sample HPB CD3 F CD4 CD45 CD3 Count Volume pL of This Plot ofAll MeanCD3FITC A Mean CD45 PE CY7 A CV CD3FITC A 0 0 Events pL 00 8 of 10 000 000 Events Gated on P1 in all This Plot 102 719 0 0 100 0 44 8 23 071 3 38 996 7 65 7 Ri 99 183 0 0 96 6 43 3 23 875 3 40 270 2 62 0 Q1 UL 21 694 0 0 21 1 9 5 540 5 32 690 2 45 5 Q1 UR 77 625 0 0 75 6 33 9 30 356 9 42 417 0 30 7 Q1 LL 3 380 0 0 3 3 1 5 447 0 1 141 0 76 5 is Q1 LR 20 0 0 0 0 0 0 8 449 7 2 092 6 97 0 ma as r a r n j R Is ii Untitled Workspace fa Microsoft PowerPor m Collect Tab Directions wE RE 5 32PM Figure 3 34 Gate for Plot 2 CD3 FITC vs
86. late Median Recalculate All Medians If a marker or region for which a Median value has been calculated is moved the Median will not automatically recalculate The notation tod to be determined will appear in the Statistics Table Figure 5 9 Recalculate All Medians should be selected to update the value Figure 5 8b The first statistics line will always read NA There is no calculation of the All row LA Median CD3 FITC A Median FL2 A Figure 5 9 a Statistics table after moving Quadrant markers www AccuriCytometers com Page 45 The Instrument Menu L5 Untitled Workspace CFlow File Edit Display Instrument About Set threshald le Set compensation Collect Run cleaning fluid cycle r Run decontamination fluid cycle ar fe Run unclog cycle Run backflush cycle Clean tlow cell C1 C2 Calibrate fluidics Update firmware Ei E2 ES EF ES EG EF E88 9 E10 E11 E12 Fi F2 FS F4 FS FR F7 Fe F9 FAQ F141 F12 G1oG2 63 G4 65 Gh GF G8 Gg 610 611 612 re nrmo Apn p z a hI Hi H2 HS H4 HS HG H7 HS Ha H10 H11 H12 Figure 5 10 The Instrument Menu Set Threshold Opens the Threshold dialogue box Figure 5 11 This allows for selecting the trigger channel setting the Primary Threshold value and setting an optional Secondary Threshold Changing the Primary Threshold to a parameter other than FSC H will change the parameter that is being used to trigger the data collection The t
87. lete and before the next well begins to acquire data Modifying Settings After a set is created its settings can be modifying by reselecting the well that are to be changed After changes are made and settings applied selected wells will change to a new color View Sample Settings After settings have been applied to a workspace the settings of a particular well can be viewed by Ctrl Click on the well of interest The red box will move to the well that has been selected this way Remove Settings After settings have been applied to a set they can be removed using the Remove Settings button Settings can be removed from one or all of the samples within the set by either reselecting the entire set double clicking one of the samples within the set or selecting the samples individually by clicking on them When settings are removed from a sample the sample well will revert to a white box The acquisition settings displayed in the Control Panel will not change Agitate While running from Auto Collect CSampler can automatically perform plate agitation at periodic intervals Agitation can be set to occur in 1 minute intervals up to 30 minutes throughout the duration of the plate run Agitate will not interrupt data collection If the interval is reached during acquire the well will finish and then the agitation will occur As stated previously Agitate can be selected to perform between 0 and 10 agitate cycles Agitate settings are applie
88. mple lits Permanently el Unlim F5C H bd less These settings will also be applied as you collect additional samples Cancel Close 30 __ OPTIONAL SECO ngate Permanently el v less Mir Apply to Only this sample Apply C All samples A Figure 3 4 Warning message when raising a threshold value will result in data loss Before collecting the first sample place an empty sample tube on the SIP and click the Backflush button lower left side of CFlow template This will clear any residue from the SIP Gently re suspend the cells in the sample tube and place on the SIP Click the RUN button to start sample collection The green Traffic Light will turn yellow and the message will read Preparing to analyze sample Once the fluidics initialization is complete the Traffic Light will turn back to green and the message www AccuriCytometers com Page 19 will read C6 is collecting data Well A1 will flash blue during data collection 13 After the run limit is reached well A1 will stop flashing and remain blue indicating that the well contains data The RUN button will now read ADD TO Figure 3 5 If desired more data can be collected into well A1 by clicking on the ADD TO button Even after collecting samples in other wells any well containing data can be returned to and more events collected at a later time Liy HPE Unetained A1 CFlow FoR Fide Edit Display Instrument About CFlow
89. mple1 and Sample 2 10 000 10 000 m Control CJ Sample 1 I Sample 2 Count 5 O00 Count 5 000 Original Data VirtualGain Applied to Data Figure 6 1 When to use VirtualGain www AccuriCytometers com Page 49 Applying VirtualGain 1 Open CFlow 2 Select the Analyze tab 3 Recreate the histogram or copy plots from Collect for application of VirtualGain Figure 6 2 VirtualGain can only be applied to one parameter at a time and must be done on a histogram plot After VirtualGain is applied the data can be viewed with or without the VirtualGain in any type of plot FR Virtual Gain Tutorial DF Fis Felt Cyl Irei Alsou OfFlow Plus Analyze Ao eo oy i a aan AG en ee ee 1 63 Ch ce ci d c ch te Cece i b Sada ee DE ILit at TI j TE Phe eo E te oi E NN ET Select a piot and E rg rji ea rg me ry a e EA ee i sample to analyze TI E ga te Ui a i a a ae z J ee oe H E a H H Make anewplot jj bil GOPY PLOTS FROM COLLECT Plot List Piok 17 Faa 8 Select a plot and Select a plot and Select a plot and Select a plot and sample to analyze sample to analyze sample to analyze sample to analyze Figure 6 2 Recreate the histogram or copy plots from Collect 4 Apply the appropriate gating to the plots in Analyze Gating is not automatically carried over in plots copied from the Collect tab 5 Click on the histogram parameter label to reveal the Parameter List Figure 6 3
90. n CFlow including the creation of complex gates setting fluorescence compensation and generating statistics In this chapter a four color analysis of human peripheral blood will be used to illustrate many of the Collect tab functions including e Acquiring data on the C6 e Creating plots histogram dot or density in which to view data e Setting stop criteria and thresholds e Using regions and markers to create gates and obtain statistics e Saving and printing plots and data A CFlow file containing the data used in the following example HPB 4 Color Tutorial c6 is available from the Accuri website www AccuriCytometers com and can be used as a tutorial tool For the example shown here the C6 has already been started up and instrument performance validated The steps outlined here follow a workflow process where the C6 user is setting and applying gates opening plots and applying fluorescence compensation values in real time as data is being collected An alternative approach is to collect all data using minimal C6 set up with only the appropriate Thresholds Fluidics rate and Run Limits set initially The data for all samples is then collected in quick succession and analysis performed at a later time including setting of gates and applying fluorescence compensation Experimental Design of Tutorial File Four tubes are used to assess the CD3 CD4 and CD3 CD8 cell populations in the example cell analysis These sample
91. n be viewed Plots can only be modified deleted or added from the Manual Collect tab Alternative Pause Commands In place of the traditional PAUSE button found in CFlow Plus CFlow Sampler contains an INTERRUPT PLATE button and an ABORT WELL button These buttons will only be active when CFlow Sampler is acquiring data INTERRUPT PLATE will complete data collection from the current well and perform specified wash and agitate cycles before stopping the plate run At this point the plate can be ejected When a plate is interrupted the Auto Collect tab is disabled except to restart and the settings cannot be changed In order to collect data during an Interrupt state the Manual Collect tab must be used This ensures that the run settings of the interrupted plate are retained when the plate run is restarted The plate run can be www AccuriCytometers com Page 65 restarted by selecting the AutoRun button Upon restart of the plate CFlow Sampler will begin at the next sample well ABORT WELL will immediately stop data acquisition stop the fluidics and eject the plate This is similar to PAUSE in CFlowPlus Upon restart of the Run CFlowSampler will begin at the next well It will not continue to collect from the aborted well Similar to INTERRUPT PLATE the Auto Collect tab is disabled after an aborted well Settings cannot be changed from the Auto Collect tab in the aborted state Acquiring Data To begin data acquisition click the AUTOR
92. nclude 1 OA OVS TNS oeeie EE R EEE EAER 22 Figure 3 11 Plot 2 has been gated to include P1 all events ce cecseeceeeceeeeeeeeeeeeeeeeeeseeeeeeeeseeeeeeeeeas 23 Figure 3 12 Gating choice options for plots Include Exclude and Intersection cccceeeeeeeeeeteee 23 Figure 3 13 Adding Vertical or Quadrant Markers to the Gating list ccccceeeeeeeeeeeseeeeeeeeeeeeeeeeeeeeeeeeeas 23 Figure 3 14 Selecting to display the 1st decade of data on a default log data plot a Open the Plot Spec dialog box b Uncheck Hide 1st decade boxes for FL1 A and FL2 A and set the Min Value for both Parane a le 0 Renee nett ee eer ee ener en er ee ee ee ee ee eer 24 Figure 3 15 A CFlow Workspace with 5 added 2D fluorescence plots Plots required for tutorial FL1 A vs FL2 A FL1 A vs FL3 A FL2 A vs FL3 A FL4 A vs FL8 A FL1 A vs FL4 A 00 ececeeeeeeeeeeeeeeeeeeeees 24 Figure 3 16 Sample number 2 has been added to well A2 in the CFlow Workspace cccssseeeeeeeees 25 Figure 3 17 Changing axes labels using the axis label drop down menu and the Rename Parameters PUNO ies ec epee sea nasa ater ea ea aa ca stance Pease eds na gece wana A oa ea inna Enea vanionclannega ea sanamanannnustanoniae 25 Figure 3 18 Changing axes labels using the axis label drop down menu and the Rename Parameters option Note that once the label CD45 PE Cy7 has been entered it will now appear in the drop down menu when the Select
93. nd for the forward scatter of the beads to make it easy to determine if the C6 performance is stable In Figure 1 8 the C6 in question had been performing with a FSC H CV of between 2 and 2 5 for over a month using the SPHERO Rainbow Calibration Particles However on the last day shown the FSC H CV had increased substantially as had the top peak CVs for FL1 H and FL2 H This could indicate either bead degradation or a slightly dirty flow cell Refer to Troubleshooting for possible fixes Also refer to Troubleshooting if any of the following conditions are experienced 1 Very broad or multiple peaks for FSC H on the 8 or 6 peak beads excluding the shadow doublet population or Fewer than 8 peaks visible on FL1 H and or FL2 H or fewer than 6 peaks on FL3 H or Brightest peak CVs are gt 5 0 in any channel www AccuriCytometers com Page 11 EJs pk Bead File 1903 CFlow File Edit Display Instrument About Clow Plus Plot 1 Sample 110808 Gate No Gating R1 85 4 Display Plot Preview Sample Selector Add rows to your master statistics table by selecting samples Add to Preview Table Sample Name C m 092908 O Vv o93008 C v 100108 C v 100208 v 103008 M iF 10508 ce MV 110808 C v 111108 Statistics Statistics Column Selector Add columns to your master statistics table by selecting a cell
94. ng the Auto Collect tab data can be collected either horizontally or vertically through the available wells starting at well A1 Naming a Plate In addition to naming individual samples wells within the plate the entire plate can also be named for www AccuriCytometers com Page 57 future reference Simply type a label in the Plate Naming Field Figure 7 1 The Manual Collect Tab The Manual Collect tab has a similar layout to the Collect tab in CFlow Plus Figure 7 3 and is used in the same manner Simply select a well as indicated by the red outline input the desired acquisition parameters and click RUN See Chapter 3 for more details Ej untitled Workspace CFlow File Edit Display Instrument About Cfrlow Sampler Plate Plate Type 96 wel plate _Eject Plate Load Eject Plate Name Clickhere to name plate A1 C1ick Here To Rename Sample 1 2 3 4 5 6 7 8 9 10 11 12 Jat az aal aal asl AB AT AB AD A10 A11 A12 SSC A B1 B2 BS B4 B5 BG BF BS BI B10 B11 B12 C1 C2 C3 C4 cS cei e7 e cai cg C10 C11 C12 Di D2 D31 D4 DS DB D7 DS DY D10 B11 D12 E1 E2 E3 E4 5 E6 EF E 8 E9 E10 E11 E12 Fl F2 F3 gt F4 FS FG F7 i FS FO F10 F117 F12 G61 621 G63 64 G5 GB G7 G8 69161016111612 ae fen an tan f fee fee bo H1 H2 H3 H4 HS HG H7 HS H9 H10 H11 H12 C6 and CFlow are connected e and ready Run Limits J Run Unlimited Flow Rate 14 pLimin E i0000 events Core Size 10
95. ocess is commonly referred to as color compensation or fluorescence subtraction 1 For each 2D fluorescence plot choose the Quadrant Tool and click inside the plot to set the quadrant position Figure 3 22 PIU fr SSPE OO CUI Pas sama GATE Flin all Fa E E a3 me E E a a a Quadrant a al e Tool Figure 3 22 Selection and placement of Quadrant Marker www AccuriCytometers com Page 27 E Plot 3 Sample HFE CO45 GATE F1 in all E E q a a a o a E b Figure 3 23 a Placement of Quadrant Tool for Median calculations on the double negative lower left and single PE Cy7 positive upper left populations b Median fluorescence values in the Statistics Table for Plot 3 Q1 will all read NA until Calculate Median is selected 2 On Plot 3 FL1 A vs CD45 PE Cy7 plot adjust the Quadrant Marker position as shown in Figure 3 23 so that the double negative and single PE Cy7 positive populations are cleanly separated into the lower left and upper left quadrants respectively 3 The Q2 quadrant marker and associated text in Plot 3 should appear bold as in Figure 3 23 If not click on any Q2 quadrant line to make it bold 4 Select Display Calculate Median from the drop down menu Figure 3 24 E Human PB CFlow Fie Edit Display Instrument About Events Display Settings fe Remove All irtualGain Calculate Median Recalculate All Medi
96. ontact Accuri Technical Support www AccuriCytometers com Page 69 Accuri Cytometers Inc Accuri Cytometers Europe Ltd 173 Parkland Plaza 56 Edison Road Ann Arbor MI 48103 St Ives USA Cambs PE27 3LF UK 1 734 994 8000 44 0 1480 308380 Fax 1 734 994 8002 Fax 44 0 1480 308381 Accuri CFlow C6 Flow Cytometer and Science is hard Flow cytometry should be easy are registered trademarks of Accuri Cytometers Virtual Gain and CSampler are trademarks of Accuri Cytometers 7820011B 2009
97. ottom of the dialog box Note that the label CD45 PE Cy7 will now become a selectable option to use for naming FL3 in other sample wells Figure 3 www AccuriCytometers com Page 25 18 i Rename Parameters x 1 Rename FL1 to FL1 none v Rename FL2 to FL2 none v Rename FL3 to CD45 PE Cy FL3 Select v Rename FL4 to FLA Be m CD45 PE Cy Apply to Sample HPB CD45 PE Cy 7 OK Cancel All samples Figure 3 18 Changing axes labels using the axis label drop down menu and the Rename Parameters option Note that once the label CD45 PE Cy7 has been entered it will now appear in the drop down menu when the Select option is chosen Naming and Saving a CFlow File Up to this point in the example the data in wells A1 and A2 are saved only in the computers RAM The data will now be stored on the computer as a CFlow File 1 Choose File Save CFlow File from the drop down menu in the CFlow command line Figure 3 19b enter an appropriate file name in the text box that opens Figure 3 19a and click Save The file will automatically be given the extension c6 indicating that the file has been saved as a CFlow data file FA HPs Auto CD45 A1 A2 plots CFlow I a I a x re Save in E Collect Tab Directions 2 El Open CFlow File or Template Pe HPB Al AZ A3 A4 Comp Analyze c Wena athe ie f E HPB Auto CD45 A1 A2 Comp c My Recent Save CFlow File
98. ows for multiple washes to be performed automatically after each well is complete Up to 5 wash cycles can be selected Experience has shown that running one wash cycle will typically reduce carry over to lt 1 0 Running two wash cycles will typically reduce carry over to lt 0 1 Agitate Clicking the Agitate button will activate a single agitation cycle Agitate is designed to keep a sample in suspension It is not designed to re suspend a completely settled sample Selecting Agitate from the Manual Collect tab will perform a single agitation cycle Using Agitate from the Auto Collect tab allows for multiple cycles up to 5 to be performed automatically at given time intervals during the plate run Agitation is only performed when the SIP is clear of any well or tube Fluidics Commands Since the SIP is not easily accessible with the CSampler installed some of the fluidics commands have been modified Backflush When the Backflush command is selected the CSampler will automatically move the arm and plate platform to the Eject position allowing a container to be placed under the SIP After placing a container under the SIP to catch any fluids that will be ejected from the SIP click the Backflush button in the Run Backflush Cycle window Figure 7 4 Run Backflush Cycle Place appropriate container under SIP to catch Fluid Cancel Figure 7 4 The Run Backflush Cycle window Unclog When the Unclog command is selected the
99. pply Settings 10 Define the file name for Autosave 11 Enter sample names and labels into the Sample Annotation Table manually or import them from a spreadsheet program 12 Select agitation schedule 13 Select the direction the plate is to be Run 14 Open the Run Display 15 Select the Plot to view during acquisition from the Plot dropdown 16 Click AutoRun 17 When the plate is complete close the Run Display 18 Switch to either the Manual Collect or Analyze tabs to review and or analyze the data www AccuriCytometers com Page 68 Collisions and Alignment If instructed to do so by the software an alignment should be performed by selecting Align from the popup Fig 7 14 Alignment can also be performed at any time by selecting Align from the Instrument Menu If there is an obstruction in the path of the CSampler arm CFlowSampler will indicate that a collision has occurred The message box shown in Figure 7 14 will be displayed together with a red Traffic Light In order to continue using the CSampler an alignment must be performed Before running an alignment any objects previously placed on the CSampler mat should be removed x CSampler has detected a collision You must align the CSampler to continue collecting This may take a Few minutes Align Figure 7 14 Collision detected window If a second collision occurs a second alignment will begin automatically If the secondary alignment fails please c
100. r left corner of each plot you want to print and then select File Print Selected Items from the drop down menu The corresponding Statistics table for each plot will automatically be selected as well These can be de selected with a click if desired Figure 4 9 File Edit Display Instrument About Open CFlow File or Template m New CFlow File gt Save CFlow File COO 4 Save as Template Import FCS File Plot 1C Sample Unstained Plot 3C Sample Unstained Plot 6C Sample Unstained GATE No Gating oat z GATE P2 in all A a GATE R2 in P2 in al 7 8 9 10 11 12 AF AS AQ AID A11 A12 Export FCS File Export ALL Samples as FCS B7 BS B9 B10 B11 B12 Print Selected Items c7 cs cg c10 C11 C12 D7 DS DS B10 B11 B41 E1 E2 E3 E4 E5 E6 EF E8 E9 E10 E11 E Fi F2 FS F4 FS FG FF FS FO FIO F114 12 61 62 63 gt 64 G5 GB G7 G8 69 G10 GY G12 E F G H H1 H2 H3 H4 HS H6 H7 HS HO H410 11 H12 a e n A a SO Check marks indicate Plots and Statistics Plot 1C Sample HPB CD Plot 3C Sample HPB CD Plot 6C Sample HPB CD Tables are selected for GATE No Gating o GATE P2 in all y GATE R2 in P2 in al printing Ps CD45 PE Cy7 A a2 200 000 400 000 590 477 FSC A ooL ZAR all 230 169 100 0 100 0 420 020 6 110 626 5 69 9 163 2 P2 101 071 00 43 9 43 9 252 819 1 27 438 6 16 2 37 8
101. rument About Cflow sampler Plate Type Field paete innr Elect Plate F Plate Naming Field p Pitename A1 Click Here To Rename Samp 1 2 3 4 5 6 7 8 9 10 11 12 A A 2 4 B z 2 7 C E 2 D 5 Select a 2 E E 2 E Plate Type G 2 H H 2 C6 and CFlow are connected e and ready Figure 7 1 CFlowSampler start up screen To assign a plate type select the Plate Type Menu by clicking on the Plate Type Field A drop down menu will appear with the options 96 well plate 96 well deep well plate 48 well plate and 24 tube rack Figure 7 2 F4 untitled Workspace CFlow File Edit Display Instrument About Crlow Sampler Manual Collect Plate Type v Eject Plate E identity Flate lype Plate Type Plate Name f E Options A1 48 well plate 24 tube rack 1 5 9 10 11 12 A A 2 SSC A B B 2 Figure 7 2 The Plate Type drop down menu The Accuri CSampler is compatible with 96 well plates standard flat u and v bottom 96 well deep well plates 48 well plates and standard 12 x 75 mm tubes in a 24 tube rack Once a plate type is selected the sample grid will display the available wells For a 96 well plate all 96 wells are available for a 48 well plate wells A1 F8 are available and for the 24 tube rack wells A1 D6 are available for data collection Data can be collected into any well and in any order using the Manual Collect tab When usi
102. s or tube rack Auto save CFlow Sampler automatically saves sample data after it is acquired Auto save will occur at the completion of data acquisition in each well whether using the Manual Collect tab or the Auto Collect tab Auto save will only save the most recent data collected If changes are made during analysis of previously collected data such as changing the threshold value these changes are not automatically saved In addition changes to analysis must be saved using the Save command from the File menu Export Sample Settings Accessible from the File menu sample settings can be exported as a csv file allowing information to be opened in spreadsheet programs Sample settings include acquisition criteria sample names parameter names and compensation values CFlow Plus Compatibility CFlow Sampler files are compatible with CFlow Plus software CFlow Sampler files utilize the same c6 extension All data and plots will be recreated in CFlow Plus While sample settings such as Wash and Agitate will not be accessible they are not lost if files are opened in CFlow Plus and then reopened in CFlow Sampler www AccuriCytometers com Page 56 Choosing a Plate Type The first action that should be performed when setting up an experiment is to select a plate type as shown in Figure 7 1 Every time a new CFlow Sampler workspace is opened a plate type must be selected PJ untitled Workspace CFlow File Edit Display Inst
103. s were prepared by staining peripheral blood with directly conjugated antibodies followed by red cell lysis according to standard methods Isotype Isotype Isotype Isotype Isotype CD45 Isotype Table 3 1 Experimental design for analysis of CD3 CD4 and CD3 CD8 cell populations in human peripheral blood Samples 1 and 2 are the background control and the white blood cell gating control respectively Sample 3 is the control required for determining the percent of CD4 and CD8 cells within the CD45 CD3 subset This experimental design does not contain single stained fluorescence controls Based on previous experiments it is known that the only channels where fluorescence bleedover introduces ambiguity for setting gates are FITC bleedover into PE and PE Cy7 bleedover into the FITC and PE channels Therefore these samples are sufficient for this tutorial Refer to Accuris online Fluorescence Compensation Tutorial for a more complete discussion of www AccuriCytometers com Page 17 fluorescence bleedover issues in flow cytometry and the impact it has on experimental design Opening a New CFlow Workspace and Collecting a Data File 1 Double click the CFlow software icon on the desktop or if CFlow is already open select File New CFlow File from the CFlow file drop down menu 2 The Default template is shown in Figure 3 1 Note that only a density plot of linear FSC A vs linear SSC A is displayed The plot is already zoom
104. s will be reused in the same order starting with the 13 set created ES Sampler Test Save CFlow File Edit Display Instrument About Cflow SAMPLER Auto Collect Plate Type a6 well plate v Eject Plate Plate Name t Select All Ctrl click to view sample settings Deselect All 1j 2 3 4 5 6 71 8 9 J10 11j 12 alja a2 as as as as ar a ag ato at anz B 561 B2 B3 B4 B5 BB BF BS B9 B10 B11 B12 C7 cs C9 C10 C11 C12 D7 D8 f D9 D101D111D12 E7 E8 E9 E10 E11 E12 FF FS FQ F10 F11 F12 G7 68 69 gt 610 611 612 H H8 H9 H10 H11 H12 l C6 and CFlow are connected and ready Run Limits Fluidics Iv 10000 events Slow Medium Fast a Flow Rate 14 pLimin it in Ungated Sample Core Size 10 um a fo Min 0 Sec Cat Thrachalad Figure 7 10 The Auto Collect tab displaying two sets The first time Apply Settings is selected CSampler will prompt a Save File dialogue box This will designate the name and location of the file that will contain the data about to be run in this session of www AccuriCytometers com Page 63 CSampler This is where Autosaved data will be saved Once the file has been saved the OPEN RUN DISPLAY button will become active Every time Apply Settings is clicked while defining the acquisition parameters for the plate the settings are resaved Once the Auto Collect Run has been started the data will be automatically saved after each sample is comp
105. seeeeeessaaegeess 44 Figure 5 7 a Quadrant marker is not selected b Quadrant Marker selected bold lines 0008 45 Figure 5 8 a Statistics table before selecting Calculate Median b Statistics table after selecting ACS Medi Mess eerste ete ace secroctsce E E E E 45 Figure 5 9 a Statistics table after Moving Quadrant markers cccccceeeeeeeeceeeeeeeeeeeeeeeeeseaeaeseeeeeeeessaeaeeses 45 FIGUIC 3210 PRE INSTFUIMGNTIWICTIU seiirsrorni a a 46 Powe oe 1 eshda ke TNS adecaneo ne secnaeceacm aie E acunstaunessaoseeesianescnaseaenes 46 Figure 5 12 Warning displayed after a change in threshold SettingS cccccecsseseeeeseeeeeeeeeeeeesseeesseeees 47 Figure 5 13 COMPENSATIONS SEUNG S acs tereeucccace custiexuccentaccdonciccarancasawecuassteuegnseecentnlboecidastaxuccunteiecewlnamecmaceaenes 47 Figure o ta TAG ADOUT MENU secesi a a a a eeseovstesienesasseencne 48 Figure 6 1 When to use VirtUalGain ccccccccccccecseseeeeeeeeeeeeeeeeseeeeeeesseesseeeeeeesessaasceeeeeessaaesaeeeeeeeesssaaaaeses 49 Figure 6 2 Recreate the histogram or copy plots from Collect cccsseseeeeeeeeeeeeeeseeeeeeeeeeeasseeeeeeeeeaaaaas 50 Figure 6 3 Click on the histogram parameter label to reveal the Parameter list sssseeeeeeeeeeeeeeeees 50 Figure 6 4 Step 7 and Step 8 Aligning plots 2 0 eee cccccccecccsesseeeceeeeeaeeeeeeeeeeeeseeeesseeeeseesseaseeeeeeeeesssaeagsees 51 Figure 6 5 Step 9 Pick the sample to align
106. t Information Opens a dialogue box containing specific information regarding CFlow and the C6 In the event of a problem with the C6 Accuri Cytometers Technical Support may ask for this information Get C6 Internal Log Disabled at this time www AccuriCytometers com Page 48 Chapter 6 VirtualGain VirtualGain is a feature in CFlow that allows the mimic of detector voltage adjustments It is strictly an analysis tool and should not be used while collecting data There are certain instances where the position of a given peak in different samples should be the same or at a specific channel number regardless of the staining Instruments that have voltage and amp gain controls allow the movement of peaks from sample to sample facilitating this CFlow has VirtualGain instead of these controls This tool allows you to mimic voltage and amp gain adjustments to reposition data on the axis after the data has been collected VirtualGain is a gross adjustment an approximate visual shift of the data In Figure 6 1 the negative peak in the Control and Sample 1 fall in similar channels MFI 28 2 and 29 7 respectively However the negative population in Sample 2 is further to the right MFI 73 4 In order to align the negative population peak of Sample 2 with the Control use VirtualGain Control Sample Sample 1 Sample 2 10 000 10 000 10 000 Negative Peak Count O00 Count 4 O00 Count O00 Overlay of Control Sa
107. t from the C6 Materials Needed to Operate and Maintain the C6 Sheath fluid Deionized filtered water 0 2 um filter plus Bacteriostatic Concentration Solution Accuri Part KR 220 Decontamination Solution 10X Concentrate Accuri Part KR 200 Cleaning Solution 10X Concentrate Accuri Part KR 210 C6 Flow Cytometer Maintenance Kits which include replacement Fluidic Bottle Filters Accuri Part CP 130 and CP 135 In line Sheath Filters Accuri Part CP 140 and Peristaltic Pump Tubing Accuri Part CP 105 Validation beads refer to page 7 System Cleaning and Validation Following a few simple rules ensures C6 optimal performance Keep the C6 Clean Always follow the startup and shut down procedures outlined in the C6 Flow Cytometer Instrument Manual in order to keep the SIP and flow cell clean and the fluidics system free of air bubbles Decontamination fluid is run through the C6 fluidics system at each shut down This helps to keep the SIP and flow cell free of debris and protein build up but may not prevent build up from occurring Always leave a tube containing at least 2 mL of filtered deionized H2O on the SIP in between uses and at shut down Replace the following User Serviceable Parts of the C6 every two months o Sheath bottle filter o Decontamination and Cleaning solution bottle filters o In line Sheath filter inside the C6 o Peristaltic pump tubing Refer to the C6 Flow Cytometer Instrument Manual Section 5 for
108. the pop up menu 9 Adjust the Marker in the FL4 H plot to be tightly placed around the top brightest peak Refer to Figure 1 7 for an example of good 6 peak bead data on a C6 10 Record the number of peaks the Mean channel numbers and CVs for the top peaks and for the forward scatter of the beads for each parameter in the Accuri Cytometer Log Additional copies can be found on the CFlow CD and on the Accuri website Please send a copy of the bead file from the first time beads are run on the C6 after installation to Accuri Technical Support dmack accuricytometers com www AccuriCytometers com Page 10 m 7 Daily Bead Analysis from Bead Template 172 7 Crlow Ea File Edit Display Instrument About Clow Plus accuri Collect E1 6 Peak Beads 2 9 09 Plot 1 Sample 6 Peak Be Plot 2 Sample 6 Peak Be Plot 3 Sample 6 Peak Be GATE No Gating GATE R1 in all GATE R1 in all 12 3 4 3 6 7 98 9 011 2 A2 AS A4 AS AG AF AB AQ A10 A11 A12 B1 B2 B3 B4 BS B B7 B BY B10 B11 B12 C11 C2 C3 C4 C5 ceg e7 i C8 co 010 011 C12 Di D2 D3 D4 DS DB D7 DS DY D10 D11 012 E E2 E3 4 E5 E6 EF f E8 E9 E10 E11 E12 F1 F2 gt F3 F4 FE FG FF FS FQ FIO F411 F12 611621631641651 GB G7 G8 G9 610 611 612 zsa7T moonown7 gt H1 H2 H3 H4 H amp S HG H7 H8 H9 H10 H11 H12 C6 and CFlow are connected and ready Plot 4 Sample 6 Peak Be Plot 5 Sample 6 Peak Be Plot 6
109. threshold setting etc Sets can be created in several ways 1 Individually select the wells 2 Click on the Column 1 12 or Row Header Labels A H This will select the entire column or row Multiple rows and columns can be selected simultaneously 3 Selecting all the sample wells in the Sample Grid Above the Sample Grid are Select All and Deselect All commands in blue text Clicking Select All will select all the wells Samples can be unselected by using the same methods An entire set can be selected by double clicking on any of the sample wells within that set Once single or multiple wells have been selected settings can be applied This is indicated by the Apply Settings button becoming active Figure 7 8 Untitled Workspace CFlow File Edit Display Instrument About Cfrlow sampler Auto Collect Plate Type a6 well plate Eject Plate Plate Name Select All Ctrl click to view sample settings Deselect All A a2 asl aal asl al a7 AB ao lato Aat A12 B1 B2 B3 B4 BS B BF BS BY B10 B11 B12 C14 C21 C3 C4 C5 C6 CF csi C9 010 011 C12 Di D2 D3 D4 DS D61 D7 DS D9 D10 D11 D12 E1 f E2 f E3 E4 E5 f E6 EF f E8 f E9 E10 E11 E12 F1 f F2 gt F3 F4 FS FG F7 E FS FO FIO F114 F12 61 62 G63 G64 G5 GE G7 G8 69 G10 611 612 H1 H2 H3 H4 HS HG H7 HS H H10 H11 H12 C6 and CFlow are connected and ready 1 2 Se SSeS SAS eee Figure 7 8 Selecting
110. ts Core Size ym a o FL1 0 00 Yo FL2 0 00 FL4 0 00 a 3 Apply Settin gs a Correct FL4 by subtracting a percentage of ao to ay TE 0 2 g FLi 0 00 FL2 0 00 FES OG One or to All Bey Bey La EE Apply to Sam pl es Sample 42 Preview Save amp Cancel amp Close Close All samples ao FIZA FIFA FL ook ses A ist o fe sree J ount Volume pL ean CD45 PE Cy7 A CV FL1 A CV CD45PE Cy7 A Median FL1 A M 01 660 0 0 1 Open Color 42 578 8 32 7 35 8 NA Set Color Compensation a 00 Co mpensation 44 700 5 27 9 27 6 841 0 gt a 0 0 0 0 0 0 0 0 0 0 0 Cumulative Delete Sample Data 4 921 0 0 Dialog box 887 2 84 8 71 3 240 0 ms aon 2 0 0 1 145 51 27 7 15 4 10 200 0 Figure 3 26 Opening and using the Compensation Settings dialog box 7 Click on the FL3 button in the middle of the top row under the words Correct FL1 by subtracting a percentage of in order to input a compensation value www AccuriCytometers com Page 29 Plot 1 Sample 42 GATE No Gat 1 Enter subtraction value Compensation Settings for Sampl A2 Correct FL1 by subtracting a pe tage of FL2 0 00 FLS 1 35 fo FL4 0 00 J ii Correct FL2 by subtracting a percentage of bile fo 00 Yo Correct FL3 by subtracting a percentage of Fld 0 00 Correct FL4 by subtracting a percentage of FEI F Yo FL2 0 00 Apply to P
111. ughout data collection and analysis there is no need to close it when moving between samples making or changing plots and applying gates or even when moving between the Collect Analyze and Statistics tabs Run Cleaning Fluid Cycle Runs a cleaning fluid cycle This cycle takes 16 minutes to complete and is different than the Decontamination Fluid Cycle that occurs during shut down Run Decontamination Fluid Cycle Manually runs the Decontamination Fluid Cycle that is performed automatically during a normal shut down of the C6 Run Unclog Cycle Runs the Unclog cycle This command is also directly available from the Control Panel via the Unclog button Running the Unclog Cycle will cause fluid to drip out of the SIP Be sure to remove your sample tube and place a blotter or empty sample tube under the SIP to catch the effluent Run Backflush Cycle Runs the Backflush cycle This command is also directly available from the Control Panel via the Backflush button Running the Backflush Cycle will cause fluid to drip out of the SIP Be sure to remove your sample tube and place a blotter or empty sample tube under the SIP to catch the effluent Clean Flow Cell The flow cell will fill completely with cleaning solution aspirated from the sample tube on the SIP This cycle will automatically shut down the C6 with cleaning reagent that will remain in the flow www AccuriCytometers com Page 47 cell allowing the flow cell to soak When the C6
112. ure 6 6 x ___ Standard Sample _ _ Sample to Align Figure 6 6 Step 11 Move the Peak Definition Marker www AccuriCytometers com Page 52 12 Click Preview The display will now show the aligned sample with VirtualGain applied and the peak of interest will be aligned in both plots 13 If the sample alignment is not in the desired position reselect the Peak Definition Marker in the Sample to Align plot and reposition it Repeat Steps 11 and 12 Standard Sample e e M e E n FLA al Sample to Align Figure 6 7 Step 14 To align other samples exactly as the first aligned sample 14 To align other samples exactly as the first aligned sample select This sample and A sample grid will open to allow selection of additional samples If the other samples need a different amount of VirtualGain VirtualGain must be set individually for each one Figure 6 7 15 Click Apply to apply VirtualGain to the data A black asterisk will now appear under the Sample to Align plot This indicates that VirtualGain has been applied to that parameter for that sample VirtualGain is only applied to the displayed data It does not alter the FCS data The VirtualGain is recorded only in the CFlow file Figure 6 8 16 Click Close Standard Sample Al Sample to Align Apply to Only this sample This sample and AE Figure 6 8 Step 15 A black asterisk appears under the Sample to Ali
113. vert to its previous calibration automatically and will operate normally However the volumes recorded will not be optimized for the new sample volume If more precise measurement is essential try calibrating again Routine Instrument Maintenance CP 102 Replacement SIP Guard re CP 105 Peristaltic Pump Tubing Set of 2 Every 2 Months CP 130 Sheath Bottle Filters Large Pk of 5 Every 2 Months CP 135 Cleaner Decon Bottle Filters Small Pk of 5 Every 2 Months CP 140 In line Sheath Filters 2 ea Every 2 Months C6 Flow Cytometer Maintenance Kit i 1 year supply of Peristaltic Tubing CP 105 Bottle Cras Filters CP 130 and CP 135 and In line Sheath Filters Every 2 months CP 140 KR 200 Decontamination Concentrate Solution 10x As Needed KR 210 Cleaning Concentrate Solution 10x As Needed KR 220 aan Concentration Solution For Sheath Fluid s Needed Visit www AccuriCytometers com for a complete list of available parts and reagents www AccuriCytometers com Page 15 Tips on Data Collection and Analysis with the C6 The Accuri C6 is a fixed voltage system There are no voltage or amplifier gain settings to adjust The detector settings for light scatter and fluorescence are optimized and balanced during assembly The Zoom Tool can be used to magnify an area of interest In Figure 2 1 the Zoom Tool has been used to focus on the lymphocyte light scatter Figure 2 1a and surface marker fluorescence Figure 2 1b
114. yze plot There are no Zoom Tools in the VirtualGain dialogue box ee O O x Standard Sample Pick sample to align Apply ta f Only this sample This sample and HE Preview Apply Close Figure 6 4 Step 7 and Step 8 Aligning plots 8 Click on the small Sample Grid in the center of the Sample to Align plot A larger Sample Grid will appear www AccuriCytometers com Page 51 9 Pick the sample to Align to adjust the peak position in by clicking its well Figure 6 5 The grey well indicates the Standard Sample already chosen Choose from blue wells for samples to Align to the Standard Only a single sample can be chosen to align To add additional samples see Step 14 VirtualGain xj Standard Sample A 02 82 ta 05 Ma AT PA Aa MO AN A2 Bl Bz BJ Ba BS BG BF BY ES BIO B11 Biz C1 c2 Cl ca ca ce c7 ca co ciocie D1 02 D3 Da DS De OF Da D9 D10011 D2 El E2 EJ Ea ES E amp EF E E9 EO Eli E12 Samp FL Fz Fo Fa F Fe FP Fa Fa PIO F11 F12 C162 Gl oh ah a aa oy cincti cr H1 H2 HJ H4 HS HG H7 H H9 MIO M11 H12 10 000 Pick sample to align HE Apply to tf Only this sample This sample and i al ee p FLA A Preview Apply Close Figure 6 5 Step 9 Pick the sample to align 4 000 Count 10 The chosen sample will now appear in the Sample to Align plot 11 Move the Peak Definition Marker in the Sample to Align plot to the center of the peak that will be lined up to the Standard Fig

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