CyAn Quick User Guide - Flow Cytometry Core Facility (FCCF)
Contents
1. Sample List 2 Protocol List 6 Additional Menu 3 Summit Control Panel to gain access to 7 Workspace with blank histograms additional screens 8 Toolbar Icons 4 Additional Menu Summit Software Control Panel Most of the operations in Summit software can be accessed through the Summit Software Control Panel The panel is located on the left side of the screen and has a series of buttons across the top You can select each of these buttons to get information related to a particular topic Each tab contains submenus that have options specific to that menu yN m ss Histogram June 2011 2 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility Acquisition Tab The Acquisition tab allows you to set the threshold at which an event will be detected by the instrument set the event triggering parameter designate parameter names specify the data types that will be collected and set the voltage and gain to be applied to each parameter From Acquisition tab you can also set up specific sample run information and view sample run statistics lt Summit V4 3 File Edit View Acquisition Histogram Gate Workspace Instrument Tools Help 2 Alignment Workspace 1 F Celts Jil B Ew AD Oe ater Sample _ Histogram Gating Workspace Es Acquisition Sample Malachowski_16 Wawa Name Value Sample name Malachowski2. c YW PE Texas Red Lin ff PE Texas Red Lin 3 PE Texas Red Area 3 PE Texas Red Area WM PE Texas Red Log Ww PE Texas Red Log VSE DFe rr Tin SE DEF C rr Tin 4 4 k k To create a single parameter histogram double click on the X axis parameter The frame for the histogram will appear in the Workspace on the right of the screen e To create a dual parameter dot plot click once on the X parameter and twice on the Y parameter The newly created frame for the dot plot will appear in the Workspace Suggestion Create a histogram for each parameter to measure to use in order to optimise voltage settings in the next step 8 OPTIMISING INSTRUMENT SETTINGS Instrument settings need to be optimised for each experiment An unlabelled sample is required to set the correct sensitivity for each parameter used in the experiment Place an unlabelled sample on the machine In the Instrument tab set the Event rate to Low Press F2 to start acquisition Cells will begin to flow through the machine Adjust flow rate up or down until you have about 100 eps Looking at graphs determine if cells are visible Optimise FS and SS parameters to get desired appearance in FS and SS plot Use Acquisition tab and adjust FS gain up or down Double click in the Gain field Type a new value or click and drag the slider until the desired value is reached 8 Use control Z to refresh data display following settings adjustment Qu SE D
3. Theshold setting is correct for your sample Modify the threshold if necessary Take care when adjusting the gain of the trigger as this also increases the threshold value 13 Optimise the fluorescent parameters a For most fluorescent parameters select Log b Right click on the histogram plot c Select Adjust Voltage d Adjust voltage click and drag the slider until the peak is in the first log decade e Convention states that unlabelled signal should be in first log decade background signal 14 Use control Z to refresh data display Create a Gate To create a gate right click on graph and choose gating tool to draw gate To apply gate to other plots right click on gate itself and choose set gate Click once in each other plot until final plot and then click twice in final plot Gate is applied to all clicked plots October 2011 10 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility Flow Rate For all standard applications keep the CyAn Flow Rate below 22000 to ensure good quality data stricter criteria apply when analysing cell cycle talk to FCCF Staff 9 COMPENSATION In many situations where you are measuring more than one fluorochrome in flow cytometry you need to perform compensation to account for the overlap of the measured fluorescent light between the different in use parameters The next steps can be done during the set u
4. all the acquisitions Acquisition Parameters Malachowski_16 May i Threshold Z Trigger settings parameters name voltage 4 05 z 99 Fs ge i gain trigger threshold and data type w bee v aame ers MPwMAW 22 2 alss MPM AG 1 0 4 opm JP EA lo OPE MPM AW 1 0 E ASIA Alexas Red P v A iv CJPE Cyb V P jv A iv Q PE Cy7 V P X A iv Violet V P jv A iv AViolet2 v P jv A iv amp MAPC vPiVAiv APC cy7 i P iv A iv PEB I co e ttf ld E Tue qe mu e L ka ka LL kat kaj ka ka T co betel lel lle I e L Bal Sample Tab Select this tab if you want to perform compensation For a detailed description on how to do auto compensation have a look at chapter 9 Summit V4 3 01 EIE File Edit View Acquisition Histogram Gate Workspace Instrument Tools Help B1 E Alignment z Workspace 1 E x A an q GE LE EE TI ae a oo Instrument Acquisition Sample i Histogram Gating Workspace PERRE x LL EBENEN L Malachowski 16 May 08 SpectrA Y BERERESEES REI Es Sample Properties Malachowski 16 May 08 SpectrA Erom eer 16 METRE B ET NENNEN Spectr SES ER Eo Ec dl Ame 16 May 2008 anank t 372504 01h 00m 05s eMRH 103 33 eps A EE ENNEM Time MSU FARHA TERRE DARRA t FS Area H FS Log E tH San FIT
5. 0 2 32 Limit 1h 00m 5s Total Events Acq Date Unknown Sh Malachowski 16 May 08 OX J wski 16 May DB Bg fA Malachowski 16 May 08 Acq Duration 00h 00m 00s 36208 avg Event Rate 0 00 eps a 45024 R8 a 28656 j Save Path D Consumables 5 30016 E 9104 File name Malachowski 16 May 15008 gsso j Output folder Acquired samples 0 Custom keywords 64 128 192 256 SAMPLEID Malachowski_16 May dias asa Violet Lin EE Total 126 00 10 13 Total 125 00 10 31 Total 131 00 10 01 126 00 2 25 RS 125 00 2 56 R3 131 00 4 00 Es Malachowski 16 May 08 67738 72831 ies g 54248 5 33869 5 36165 EUN o 16934 o 18082 128 f gt Malachowski 16 May 08 3 gt Malachowski 16 May 08 38245 a 28683 519122 ws 9561 6 sition Parameters Malachowski 1e Maywana This Z os Trigger Fs j Voltage Peak N Peak Violet Lin APC Lin ape oT T Peak Peak Total 133 00 9 69 Total 125 00 10 20 Total 127 00 10 03 R9 133 00 R2 125 00 2 11 R10 127 00 2 20 Pasar Peak Peak lab3 Monday May 19 2008 09 34 56AM violet 1 Peak Alignment Malachowski_16 May 08 Spectra Violet 2 Peak Pc Peak APC Cy Peak liil a Ready E Bj Cyan 3 Alignment JJ Malachowski 16 May 08 Spectr i 0 0 01 1 Summit software Main Menu 5
6. 16 May 70 Spectra Sample description Limit 1h 00m 5s Total Events Acq Date Unknown cq Duration 00h 00m 00s avg Event Rate 0 00 eps Save Path D Consumables File name Malachowski 16 May j Output folder Acquired samples Custom keywords j SAMPLEID Malachowski_16 May Ali Acquisition Parameters Malachowski 16 May Threshold Z as Trigger Fs if Peak Ar Voltage Peak N Peak Peak Peak ce PESKE ua Peak Peak Violet 1 Peak Violet 2 Peak PC Peak iPC Cy Peak 4 E lili B Ready J gt cyan 1 Alignment B Malachowski 16 May 08 Spectra ey Acquisition Sample Malachowski 16 May 0g Malachowski 16 May 08 SpectrA Name Value Acquisition Sample Panel Sample name Malachowski 16 May e Numb 70 The Acquisition Sample Panel can be Em eR Operator sample description Limit 1h 00m 5s Total Events customized to display and later saved information specific to a sample run October 2011 Date Duration gs Event Rate Path gt EE name Output folder Custom keywords SAMPLEID Unknown 00h 00m 00s 0 00 eps D Consumables Malachowski_16 May Acquired samples Malachowski_16 May Version Facult des Sciences de la Vie Flow Cytometry Core Facility You can also edit
7. C Lin Kosa FITC Area H H anme FITC Log PE Log APC Log HHH u 100 0000 0 7820 0 0000 HARRAN BUNC EE ol TT l 1l Ji Ready gt cyan 2 Alignment J Malachowski_16_May_08_SpectrA 1hour NewAssy 0 0 01 October 2011 4 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility Histogram Tab Histograms and dot plots are created in the Histogram tab The Create Histograms panel displays all of the parameters that are enabled in the Acquisition tab Summit V4 3 File Edit View Acquisition Histogram Gate Workspace Instrument Tools Help IS E Alignment M oa z ular izar Acquisition Sample Histogrem Gating A Create Histograms s Plots Malachowski 16 Histogram L Malachowski_16 May n8 Spectr 1h X parameter n Y parameter 4 3 Pulse Width Q heo GZ Sm Wm 3 PE Texas Red Lin 3 PE Texas Red Area rd Texas Red Log 3 PE Texas Red Lin 3 PE Texas Red rea E Wf PE Texas Red Log j oh eed ee a i gt UU d LE Statistics Malac I amp uUnivariates _ PE Texas Red Lin L PELin viatet 1Lin violet 2 Lin violet 1 Area L aPC Cy7 Lin L PE Cys Lin aPC Area L rE Cy Lin _ Time vs FITC Lin Pulse width vs FS vialet 1 Lin vs Vio FrTC Lin vs PE Cy 4PC Lin vs APC C FS Lin vs SS Lin lt Jill
8. Click the menu icon in the top left of the actual graphs and choose Compensate 2 Adjust scroll bars to compensate samples appear Adjust the spill over percentage so that the Y axis median fluorescence value for the positive population lower right hand quadrant is equivalent to that seen with the negative population lower left hand quadrant Bi G1 R1 CD4 FITC 10 10 lt 5 ILE G1 R1 CD4 FITC_10 p Negative cell population Y axis median fluorescence p th reahic s r value Cas YelowLog Com 109 100 10 10 10 408 CD4 FITC Log Comp Positive cell population Y axis median fluorescence 10165 100 00 0 0 00 0 0 00 6331 62 29 3334 37 72 value Lower left hand quadrant Lower right hand quadrant Negative cell population Positive cell population 3 It is also possible to enter numbers by hand into compensation matrix under Sample tab 10 CLEAN UP SHUT DOWN ROUTINE Once all data has been saved it is time to shut down the instrument We have two shutdown procedures A Monday till Friday from 8 00 am to 5 00 pm B After 5 00 pm or in the weekends A Monday till Friday from 8 00 am to 5 00 pm 1 Place a 34 full tube of Decontamination solution on the machine In the Instrumenttab click Sample Clean takes 60 seconds Remove the tube and an automatic Backflush occurs Repeat the steps above with a 34 full tube of Cleaning solution and then with a 34 fu
9. E uei eh 9 October 2011 9 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility 9 Select the Acquisition tab and adjust the SS voltage up or down Double click the Voltage field Type a new value or click and drag the slider until the desired value is reached Sample Parameters Sample Parameters T Bi Threshold Z 5 4l Trigger PE Cy Threshold 5 z Trigger PE Cy7 Peak Area Log Peak Area Log Peak drea Log Peak drea Log Peak Area Log Peak Area Log Peak drea Log Peak Area Log Peak Area Log Peak drea Log Peak Area Log Voltage N Peak Area Log Peak drea Log Peak Area Log Peak drea Log Peak Area Log Peak drea Log Peak drea Log Peak drea Log Peak hrea Log Peak drea Log Peak drea Log FEF e HB HB HB FPF FF oH H K I 0O MON O NEN CO BEN C BE SOPs oO pat Pe oO fel O he FF PFP F H H H H Peak rea Log Peak drea Log 10 Aim to place main population in the central domain of FS SS graph Appearance of signals and optimisation of these parameters varies enormously between different types of samples eg mesenchymal stem cells versus murine bone marrow versus polystyrene beads will all look very different from one another and have very different FS and SS settings 11 Select FS as the Trigger To change the trigger click on the arrow and select another parameter The FS is most commonly used as the Trigger 12 Check that the
10. ETTINGS CREATE A GATE FLOW RATE 9 COMPENSATION AUTO COMPENSATION MANUAL COMPENSATION ADVANCED USERS ONLY 10 CLEAN UP SHUT DOWN ROUTINE A MONDAY TILL FRIDAY FROM 8 00 AM TO 5 00 PM B AFTER 5 00 PM OR IN THE WEEKENDS 11 DATA EXPORT October 2011 15 OO U1 U1 jl A gt CO Q N 2 N I OA OA OA OA GN S N co 10 11 11 11 13 13 14 14 Version 1
11. Facult des Sciences de la Vie Flow Cytometry Core Facility mr pf ECOLE POLYTECHNIQUE CyAn Quick User Guide FEDERALE DE LAUSANNE 1 INSTRUMENT LAYOUT 458 nm Laser s Photomultipher Tubes ma 488 10 53640 573125 61 ip Emission Filters ra 675LP 9596 SDLP 95DLP 40DLP 30DLP 7 P miror 4 z 0 _ e 4 0 53 660 20 Filter configurabons are CASB Y easily modified to CFP CAS accommodate different DAPI applications Fluorescence amp Scatter FL6 FL from Interrogation Point akV Q IOKkwIZW KWI K H I I I IIIIIIXIIIIINA T 2 635 nm Laser 405 nm Laser Figure Optical Layout of a CyAn Each group of detectors is indicated by colour Blue detectors measure light from the blue laser 488nm Red detectors measure light from the red laser 635nm Violet detectors measure light from the violet laser 407nm Detector names default and commonly used dyes and fluorophores FITC FL 1 FITC ALEXA 488 GFP CFSE YFP PE FL 2 PE dsRED PE TxRed FL 3 PI PeAlexa610 PeTxRed PE Cy5 FL 4 PeCy5 PeCy5 5 PerCP 7AAD PE Cy7 FL 5 PeCy7 Violet 1 FL 6 Pacific Blue DAPI Hoechst for cell cycle Live Dead Violet Violet 2 FL 7 Pacific Orange APC FL 8 APC Alexa 647 APC Cy7 FL 9 APC Cy7 APC Alexa750 For others dyes and fluorophores please have a look at our web site under Analyser CyAn Analysers or ask the FCCF Staff From Monday to Friday the FCCF
12. Instrument tab choose sample clean 11 Leave the machine with a tube of H2O on the sample probe 12 Turn OFF the computer Do not turn off the CyAn If you cancel your reservation after 5pm and you are supposed to be the last user of the day you are responsible for shutting down the machine 11 DATA EXPORT Data analysis of the saved files is always performed on another computers To get saved files to your own computer you can use a USB key burn a CD or use the network to connect to your share lab folder Data is removed from the computer at the end of every month by the FCCF staff Each user is responsible for backing up his data Please backup your data right after you finished acquiring your samples October 2011 14 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility Table of Contents 1 INSTRUMENT LAYOUT DETECTOR NAMES DEFAULT AND COMMON DYES USED IN EACH 2 SUMMIT OVERVIEW SUMMIT SOFTWARE SCREEN OVERVIEW SUMMIT SOFTWARE CONTROL PANEL ACQUISITION TAB ACQUISITION SAMPLE PANEL SAMPLE TAB HISTOGRAM TAB 3 LOCATION AND TYPES OF DATA STORAGE PERSONAL FOLDERS DATABASE PROTOCOL DATA FILES 4 SAVING DATA SAMPLE NAME SIZE OF SAMPLE FILE STORAGE LOCATION AUTO SAVE COMMAND COMMANDING SUMMIT TO SAVE FILES TO SAVE 5 START UP 6 CHOOSING AND NAMING PARAMETERS TO MEASURE PARAMETER ACTIVATION PARAMETER NAME 7 CREATING GRAPHS TO VIEW DATA 8 OPTIMISING INSTRUMENT S
13. chine Press F3 at the end of any acquisition that you want to save This command is also in the acquisition drop down menu October 2011 6 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility Files to save As well as saving your experimental samples save your unlabelled samples as well as any other controls eg compensation controls as these can help you to validate your data during data analysis 5 START UP 1 Make a new database and save it to your folder a Go to File menu and choose Database gt New b Name the database and click Save 2 Select the Instrument tab upper left and click Startup Event rate iv On 4 amp 9nm Opened V On 636mm Opened Startup Shutdown Backflush Vacuum AA ee Debubble Fhidice off Samp Clear Sye Clean 13068 Place a 3 4 full tube of Decontamination solution on the machine In the Instrument tab click Sample Clean takes 60 seconds Remove the tube and an automatic Backflush occurs Repeat the steps above with a 34 full tube of Cleaning solution and then with a 34 full tube of H20 7 The Instrument is now ready to be set up for your experiment ON Ur as oo October 2011 7 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility 6 CHOOSING AND NAMING PARAMETERS TO MEASURE Before data can be acquired data parameters must be enabled Parameters are chosen and
14. gs Cyan 31 Alignment J Malachowski 16 May sample Paint pur NewAssy You must create histograms and dot plots in order to display the data you acquire Prior to creating dot plots and histograms you must enable the parameters you would like to collect See chapter 7 on how to create graphs to view data 3 LOCATION AND TYPES OF DATA STORAGE Personal folders All data is stored in the data storage drive D on the Cyan s computer There is a folder for each month and each group or institute has a folder inside this folder Each user has to create a personal folder inside their group folder Database A database is essential to run Summit It contains the protocols and links to all the files that are saved Make a new database each time you begin an experiment this minimises the problems with software bugs October 2011 5 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility Protocol Save your protocols into the folder Settings for Everyone in the data storage drive D A protocol is a collection of graphs and instrument settings that you use to run your experiment inside the database These can be saved separately to the database to be re used in subsequent experiments Saving a protocol for future use is the easiest way to access settings and gates for next time Saved protocols can only be re used for experiments measuring exactly the same parameters do not add or subtract parameters from
15. ll tube of H20 5 Go to File menu and select Database and then Load select the Between Users Database on the desktop 6 In the Instrumenttab click Fluidics off 7 Place a 34 full tube of H2O remove the tube and an automatic Backflush occurs 8 The fluidic is now OFF and you can now leave the machine like this Pr qe October 2011 13 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility B After 5 00 pm or in the weekends 1 Place a 34 full tube of Decontamination solution on the machine 2 In the Instrument Tab click Sample Clean takes 60 seconds 3 Remove the tube and an automatic Backflush occurs 4 Repeat the steps above with a 34 full tube of Cleaning solution and then with a 34 full tube of H20 5 After running the water go to File select Database and then Load select the Between Users Database on the desktop Check the reservations on the SV INTRANET if there s another user after you 1 In the Instrument Tab click Fluidics off 2 Place a 34 full tube of H20 remove the tube and an automatic Backflush occurs 3 The fluidic is now OFF and you can leave the machine like this If there s no user after you 6 Inthe Instrument tab choose Shutdown 7 Place a 3 4 full tube of Cleaning solution on the machine 8 Inthe Instrument tab choose sample clean 9 Place a full tube of H20 on the machine 10 In the
16. named in the Acquisition tab Parameter activation To activate a parameter Double click in the Peak Area Log field Select the check box that pertains to the data type that you want to acquire Peak FS amp SS Linear scale Log Fluorescent antibody stains Logarithmic Scale Area Doublets discrimination To discriminate doublets select Peak amp Area for FS To Cell Cycle users please check settings with FCCF Staff Parameter name To change the name of a parameter double click the name of the parameter in the Name column type a new name and then press ENTER Sample Parameters B Threshold Z a Tii reshold Z 5 rigger PE Cy Peak Area Log Voltage Peak Area Log N Peak drea Log Peak Area Log Peak area Log 488 PE Texas Red Peak Area Log PE Cy5 Peak Area Log Peak Area Log Peak Area Log Peak drea Log Peak drea Log Peak drea Log October 2011 8 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility 7 CREATING GRAPHS TO VIEW DATA Graphs are created in the Histogram tab top panel Only fluorescent and scatter parameters chosen previously are available for graph creation Beas AY Instrument Acquisition Sample Gating Workspace 1 Histogram Tab E Create Histograms Plots Malac a i L Histogram Malachowski 16 May 08 SpectrA ih v 2 K ax i AMA X parameter Y parameter 3 Y axi S Pa ram ete rs v GEESE Lin
17. p phase during data analysis of AFTER the experiment is complete If you choose to the compensation during data analysis rather than during the set up phase it is ESSENTIAL that you save a copy of each of your single stain controls Auto compensation Before you can perform automatic compensation you have to first optimise the voltages for each of detector that you use and save a copy of each single stain control To do the Auto compensation 1 Click the Sample tab 2 In the Sample Compensation panel click the small blue icon in the upper left corner and select Auto Compensate from the list Auto Load from Sample visiComp k Auto Compensate Clear Coefficients Detach Floating Detach Printable Copy to Clipboard wv 3 The Auto Comp Sample dialog box appears Gate G1 riment Analysis 3 CD8 PE TR CD8 PE Cy5 coe PE Cy7 cmai 4 Selecta gate from the Gate list if applicable October 2011 11 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility 5 From the Experiment list select the experiment folder that contains your control samples 6 Allocated all of the single control samples to the appropriate channel Click on the arrow to select the sample 7 Click OK A new Workspace labelled AutoComp is created and the first set of dot plots is displayed Each dot plot places the control parameter on the x axis and a parameter to compensa
18. staff runs a QC protocol on both CyAn s first thing in the morning At the same time we are also taking care of emptying the waste and filling the sheath reservoir When you arrive at the platform the CyAns are turned on the summit software is running and an empty database called between users is open The machine is now ready to use June 2011 l Version 1 Facult des Sciences de la Vie Flow SHOES Core SOPHIE 2 SUMMIT OVERVIEW Summit software is a Windows R based application that has a series of menus hot keys and buttons which allow you to acquire data in FCS format With Summit software you can monitor and control the instrument define protocols configure compensation settings and workspaces Summit Software Screen Overview File glview Acquisition Histogram Gate Workspace Instrument Tools Help xu 7 Workspace 1 ns gt J ae ae Eu p ple gt Workspace gt h gt lt gt az E W m s gt Malachowski_16_May_08_ 7 GJ Malachowski 16 May 08 m 74164 66817 g 98623 g 90 12 5 37082 5 33408 18541 16704 128 128 192 PE Lin p Texas Red Lin o j Sample name Malachowski_16 May Number 70 j Source Spectra EEEE J Region Medan cv Regon Medan cv Hs ES Total 131 00 9 43 Total 129 00 9 59 Total 131 00 9 39 Sample description RS 131 00 2 07 R4 129 00 2 07 R 131 0
19. te against on the y axis Default auto compensation Dim and Bright regions are displayed and if a gate was selected it is applied to each dot plot 9 Atthe same time the Auto Compensate wizard appears Auto Compensate ompensating Einstein 13 JAN 2005 61 all 10000events control lease adjust the dim and bright regions to contain the im and bright populations 10 10 103 10 10 102 10 CD20 FITC Log Comp CD20 FITC Log Com Total 3952 100 00 38 07 1 00 10 Total 3952 100 00 38 07 1 00 10 Dim 3498 86 84 33 06 1 00 37 Dim 3455 86 84 33 06 1 00 37 Bright 9e4 13 16 5 01 140 94 Bright 924 13 16 SL 131 11 k k 10 Examine the Hist statistics for each histogram If either the Dim or Bright region contains less than 5 of the data for the dot plot click and drag the region until greater than 5 of the data appears in both the Dim and Bright regions 11 When all regions on all plots contain greater than 5 of the data click Next on the Auto Compensate dialog box and the next set of dot plots will appear 12 Repeat step 10 until all single control samples have been compensated When auto compensation is complete the compensation matrix contains the appropriate values and the AutoComp workspace is removed Compensation matrix can be inspected in bottom panel of SAMPLE Tab October 2011 12 Version 1 Facult des Sciences de la Vie Flow Cytometry Core Facility Manual compensation Advanced users only 1
20. the protocol after it has been saved Data Files FCS 3 0 files saved by Summit contain all the measured signals that you saved for any given sample They can be re opened in Summit or opened in Flowjo for data analysis 4 SAVING DATA To set up Summit to save your data make the necessary adjustments in the Acquisition tab Sample name By double clicking on top row sample name you can adjust the rules that Summit uses to name your data file A window appears with a rule string listed along the bottom Delete this and then re create your own rule string by choosing the rules that you wish to use Size of sample file The size of your file is up to you It is controlled in the limit row Double click on the number of events to edit the limit Leave maximum events at 10 000 000 Hint the size of your data file will be related to the proportion of cells you are interested in out of your total population Storage location Use Save Path to specify the folder where your files will be saved This folder should be inside a folder which should be inside your personal folder Auto save command To ensure you remember to save your files it is recommended to go to the drop down menu Acquisition and select Auto Save This makes the software request a save each time you stop acquisition ie each time you press F2 Commanding Summit to save F3 is the shortcut key to save the data that has been passed through the ma
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