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LSR II User Guide V5

1. E ECOLE POLYTECHNIQUE LSR II Quick User Guide FEDERALE DE LAUSANNE Flow Cytometry Core Facility September 2013 v 5 During normal workdays the LSR II will already be up and running and the FCCF staff will have done a performance check The software will be ready for you to log in with your personal username and password Please be aware that every month we will delete all the experiment in the software and on the computer Please make sure that you take the experiment and FCS files with you You are responsible for your own backup Only use the clear FACS tubes on the LSR II Note FACS tubes that you use on the Cyan do not work properly on the LSR II All samples must be filtered just before acquiring P2 cells have to be fixed before running on the LSRII Start up Only required if you are using the LSR II on the weekend or on public holidays 1 Turn on computer and log in 2 Turn the flow cytometer on Green button in front of cytometer The cytometer connects automatically While connecting the message Cytometer Connecting is displayed in the window footer When connection completes the message changes to Cytometer Connected 3 Turn the UV laser ON From the shortcut BD Coherent Connection program created in the desktop the program window will open Double clic i shortcut F S BD z E 3a BD Biosciences s 4 The program window will open AD Coherent Connection Hels l ar I D Genesis 39 n
2. e doublets Cytometer FACSAria 1 oO wo Parameter Voltage FSC 47 55C 52 FITC 23 PE 34 PE Texas Red 22 PerCP Cy5 5 PE Cy APC APC Cy iJ a I A 0 ojm so H a o0 oO Hal a BEJ nn kul o o o o o o o Running Samples 1 2 3 4 gt Install your tube onto the SIP Set the current tube pointer to appropriate tube Press Run on the fluid control panel Click Acquire Data in the Acquisition Dashboard in the Diva Software a Be aware that just clicking acquisition does not record any data Set the number of events to record Click Record Data a Recording will stop once the cytometer reaches the number of events to record Remove the tube from the SIP Let two drops fall down before installing the next tube This limits the carry over from one sample to the other If rear cells are of interest run some water in between the samples Attention The fluidic system is controlled by the panel located on the cytometer and not by the software If you stop the acquisition in the software the sample continues to be sucked into the machine and you will loose your cells To stop the aspiration of your sample press the standby button on the control panel of the cytometer and remove your sample from the SIP Creating Plots and Histograms Plots and histograms are created in the Global Worksheet The Global Worksheet is the main working area where you create plots define gates sho
3. ecord Data a The software automatically records 5000 evt 10 Select P1 right click and select Apply to All Compensation controls 11 Run and record each single color compensation tubes 12 Adjust the P2 gates to fit the positive populations 13 Select Experiment gt Compensation Setup gt Calculate Compensation 14 Rename the compensation with the current date and your name F Single Stained Setup Compensation calculation has completed successfully Name Experiment aj Link amp Save Apply Only 15 Select Link amp Save to apply your compensation to your acquisitions 16 If you excluded the Live Dead marker in the compensation setup a Right Click on Cytometer Settings b Select Unlink from compensation c Run and record your PI sample 17 Switch back to the global worksheet to start creating graph to view the data Global Worksheet Global Sheet1 AS IRIS LA eepal e s en DE ce Go S a ob we E Buttons to switch between compensation worksheet and the global worksheet Define labels for each parameter 1 Choose Experiment gt Experiment Layout 2 Select a label and enter the parameter e g CD3 into the label text field under the Quick Entry 3 Select the next label and enter the next parameter etc 4 When all the parameters are assigned to a label are click OK Experiment Layout Quick Entry J s o Export Experiment and FCS files 1 Select your exper
4. en 65 mW ISM THY697 X Coherent se The desired power can be typed in the power command window on set please keep this value on 30 00 mW 5 Start BD FACSDiva software by double clicking the shortcut on the desktop and log in to the software Use your personal username and password to log in to the FACSDiva software 6 Allow 30 minutes for lasers to warm up and stabilize 7 During this time run some water on low FACSDiva VVorkspace BD FACSDiva Software Administratori Ue Browser Experiment 002 xX F Global Worksheet Global Sheet1 12 19 03 10 16 14 12 19 03 10 11 00 12 19 03 10 07 06 Experiment Name Experiment 002 Specimen Name Callbnte Beads Tube Name Tube_001 Record Date Dee 19 2003 10 00 24 AM Fr SOP Administrator B Acquisition Dashboard i 55 TN MO ce 1 420b Qeca Ibec 1be 2 942 rent Activity Linear Seale 0 10 000 Tube 001 1 Browser 2 Acquisition Dashboard 3 Inspector 4 Global Worksheet 5 Cytometer Setting up an experiment 1 Use the browser toolbar to add an experiment red border Or in the menu bar go to Experiment gt New Experiment be Browser MyExperiment 2 Select Cytometer Settings in the browser B L MyExperiment 1 4 07 8 54 15 Cytometer Settings h a Global worksheets 3 In the Inspector window delete the parameters not needed a To delete parameters click the selection button nex
5. iment right click and select Export gt Experiment P Export Experiments Delete experiments after export Zip File Export Directory D BCExport Experiment Practice Experiment Date Simple_Analysis 12 20 06 3 24 10 PM A v a Note The experiment can only be opened with the DIVA software 2 Export your experiment to the D drive on the Data folder 3 To export the FCS files select your experiment right click and select Export gt FCS file a Select FCS 3 0 b Click OK c In the Save Export dialog box verify the file storage location same as Experiment In between users 1 Run 3min of Decontamination solution on high 2 Run 3min of Rinse on high 3 Run 3min of a new tube of Water on high 4 Log out from the software Last User of the day and Shutdown 5 Run 3min of Decontamination solution on high Run 3min of Rinse on high Run 3min a new tube of Water on high Shut down the software Shut down computer 0 Press the green button in front of LSRII to shut down the cytometer Broo 10 Changing the Sheath and VVaste Tank Alvvays ask staff to change the tanks for you If nobody is around Make sure that the cytometer is set to Standby before changing the tanks 1 Sheath Tank a Unscrew the lid and carefully remove sample line and put it in the holder to your left b Press the Alarm button to turn off the Alarm c Geta new full BD FACS Sheath box on the right next to the entrance d Put the lid with the
6. sample line back on the new box and put the box back to original position e Press Restart 2 Waste Tank a Remove the black holder that holds up the opening of the waste tank b Unscrew the lid of the waste tank c Get an empty waste tank from under the sink to the left of the LSR II d Exchange the lids e Put the full tank back under the sink 3 Prime the fluidics after changing the tanks a Install an empty tube on the SIP b Press the PRIME fluid control button to force the fluid out of the flow cell and into the waste container c Repeat the priming procedure d Install a FACS tube clear plastic with 1ml of DI water on the SIP and run it on high for about 2min 4 The cytometer is ready to use 11
7. t to each unneeded parameter Hold down the Ctrl key to select more than one When you are finished selecting click Delete Inspector Cytometer Settings Cytometer Settings Parameters Threshold Ratio Compensation Parameter voltage Log A FESE 250 SSC 00 FITC 500 PE 500 PerCP Cy5 5 500 On0 0 00H00000 1 4 o jon mane PE Cy v En Pacific Blue 500 Kyj K K E e E AmCyan 500 Indo 1 Violet 500 Indo 1 Blue 500 3 gt 3 IEEE 4 To change a parameter click on the name and a drop down list with all available parameters appears 5 sx Click the New Specimen button to add a specimen and a tube to the experiment D Browser Manual Comp Click the New Tube button to add a second tube To rename Experiment Specimen or Tube right click and select Rename Click to set the current tube pointer to Tube_001 in the Browse The pointer changes to green and five green A Experiment 001 tabs appear in the Cytometer window The 3 25 Cytometer Settings current tube pointer indicates the tube for a amp Global Workshaebs which cytometer settings adjustments will apply 3 and for which acquisition data will be shown 3 44 specimen 001 T Tube_001 j U Tube_002 Click the Parameters tab in the Cytometer window to check the parameters Select H and W parameters for FSC and SSC to be able to exclud
8. w statistics and population hierarchies and enter custom text Global Worksheet Global Sheett1 BAN RE E A di je S nm DEH amp ON La Buttons to create dot Buttons to create gates plots contour plots and histogram Compensation Set up 1 Select Experiment gt Compensation Setup gt Create Compensation Controls 2 Check that all the parameters that you require are listed Remove the parameter for the live dead dye this can be added again after the compensation a Other option is to kill some of the cells to ensure that there is a strong signal in the live dead channel 3 Click OK and a new specimen called Compensation Controls appears in your experiment Click on the to display the individual compensation control tubes 4 Set the current tube pointer to the Unstained Control tube under the Compensation Control Specimen E S Compensation Samples 33 ytometer Settings iki Unstained Control iia FITC Control iki PE Control ili PercP Cy55 Control iki APC Control 9 ll S eB Terte 5 Load the unstained control tube press Run on the LSR and click Acquire in the software 6 Adjust the P1 gate to include the population of interest 7 Adjust the voltage for each parameter to ensure appropriate background signal 8 You should see both sides of the histogram usually the negative signal is within the first decade of the histogram FITC Stained Control 9 Click R

LSR II User Guide V5

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