FACS Hand Outs

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FACSCalibure Flow Cytometry Hand outs Administrator: Rokhand Arvan, Dr Dennis Kasper’s lab Phone # (617)525 0744, [email protected] On Line Calendar for sign up Use the following on line calendar to book a time for using the FACS machine. Username and password will be provided upon completion of your training. http://os.med.harvard.edu/cgi-bin/microcalendars.cgi? Op=UserLogin Start up 1- Turn on FACSCalibur cytometer (on the machine, “Low” flow rate light is green & “Standby” light is orange) 2- Turn on FACStation computer (laser lamp will warm up (5 min), while you are checking the fluidics containers) Note: Always turn on the cytometer before turning on the computer when acquiring data. This enables the computer to recognize that the cytometer is connected. When analyzing data, it is not necessary to turn on the cytometer. 3- Open the Fluidics (slide drawer) to check if the “Sheath tank” contains Flow/Sheath buffer or the “Waste tank” is empty. 4- To fill the sheath tank, flip the vent valve toggle to depressurize 5- Remove the sheath metal bracket, disconnect the sheath tank connection tubes and take the sheath tank out. 6- Fill only 75% of the sheath tank. (Avoid filling the sheath reservoir to its maximum capacity, because when it is pressurized, fluid may be forced into the air supply tubing, preventing proper pressurization.) 7- Replace the sheath tank/metal bracket/Connecting tubes/detection probe connector (No kink in tube lines) 1

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Transcript of FACS Hand Outs

Monthly Maintenance

FACSCalibure Flow Cytometry Hand outs

Administrator: Rokhand Arvan, Dr Dennis Kaspers labPhone # (617)525 0744, [email protected] Line Calendar for sign up

Use the following on line calendar to book a time for using the FACS machine. Username and password will be provided upon completion of your training.http://os.med.harvard.edu/cgi-bin/microcalendars.cgi?Op=UserLoginStart up

1- Turn on FACSCalibur cytometer (on the machine, Low flow rate light is green & Standby light is orange)

2- Turn on FACStation computer (laser lamp will warm up (5 min), while you are checking the fluidics containers)Note: Always turn on the cytometer before turning on the computer when acquiring data. This enables the computer to recognize that the cytometer is connected. When analyzing data, it is not necessary to turn on the cytometer.

3- Open the Fluidics (slide drawer) to check if the Sheath tank contains Flow/Sheath buffer or the Waste tank is empty.

4- To fill the sheath tank, flip the vent valve toggle to depressurize

5- Remove the sheath metal bracket, disconnect the sheath tank connection tubes and take the sheath tank out.

6- Fill only 75% of the sheath tank. (Avoid filling the sheath reservoir to its maximum capacity, because when it is pressurized, fluid may be forced into the air supply tubing, preventing proper pressurization.)7- Replace the sheath tank/metal bracket/Connecting tubes/detection probe connector (No kink in tube lines)

8- To empty the waste tank, disconnect the waste tank connection tubes, remove the waste tank and discard the waste fluid in the sink. Caution: It is good practice to empty the waste reservoir when you fill the sheath reservoir. This prevents the waste reservoir from overflowing.

Important Note: If your samples contain hazardous material discard the waste fluid at the end of your experiment based on proper bio-safety protocols. Also, wear appropriate safety gloves/lab coat when handling waste materials.9- Add 400ml of bleach (10% final volume) to emptied waste tank and replace the waste tank/Connecting tubes (No kink in tube lines) on its position.

10- Flip the vent valve toggle to pressurize.11- Check the filter and sheath tube lines for bubbles (Remove bubbles by using the roller clamp for filter or disconnecting tubes and pressing the tip of the valve against the wall of a beaker.)

12- Enter your user ID and Password.13- Proceed to the next step (QC).

Calibration or Quality Control (QC) Test

QC is a regular test of the machine performance which will be performed by the administrator. In QC test the FACSComp program will automatically performs PMT voltage test, Time Delay calibration, Compensation, and Sensitivity test.Important Note: You dont need to perform the QC test unless the administrator asks you to do so. The QC data are saved in the QC log book as a reference.

1- For QC test, prepare two tubes, named A and B. Add 1 ml and 3 ml of sheath buffer to tube A and B, respectively. Then add one drop of each CaliBRITE beads based on table 1. (A four color setup is recommended. If no APC bead is available, perform three color setup using PerCP.)

Tube A = unlabeled + APC

Tube B = unlabeled + APC + other three labeled beads

Note: Keep the CaliBRITE beads in Fridge (4C). The bead suspensions are stable in 4C for 1 hour.

2- Open FACSComp software, enter your name as operator, click on Accept

3- Check if the beads lot ID is correct (lot IDs are printed on the box containing the beads).

4- Check that both Lyse/Wash (LW) and Summary Report boxes are marked, and click OK

5- On the Cytometer set the Flow rate on HI, install tube A, and press the Run button

6- Click on the Start, the program will automatically check PMT voltage & Time delay calibration.

7- Install tube B (mixed beads), and click on the Start, it will perform compensation & sensitivity test

8- Remove beads, install DI water, and set the machine on Standby mode.

9- A copy of QC data files will be saved in FACSComp folder if you had checkmark the summary report box.

10- Quit the program and it will print a copy of todays QC result

11- Fill the QC log book and insert the printed QC sheet in the log book.

Note: The QC can be done manually if the automatic QC does not end in 75 seconds, or the event rate is low. In this circumstance we may change numbers in Detect/Amp and Target Value window. But this is not recommended for non-admin users.Abbreviations:

Lyse/Wash (LW) = Ab is washed away in typical immuno-phenotyping protocols [in Lyse/No Wash (LNW) protocol antibody is not washed away),

HLA-B27 Calib = A clinical assay protocol,

Coated Bead assay (CBA) = An ELIZA type protocol.OptimizationTo adjust the Cytometer setting for your experiment, you need to perform an optimization test before acquiring any data.

1- Open CellQuest Pro program, it will open an untitled file for you.

2- You can save this file as an optimization file in your folder or use the Standard Acquisition Document (Std Acq Doc) which is available in the software.

3- Go to Acquire > Connect to cytometer

4- Create a dot plot (Go to Plot > Dot plot, or use the tool box to draw a plot)

5- In Inspector window for this plot, select the file type as Acquisition Analysis (The axis in the plot will change to FSC/SSC as X/Y parameters).

6- Go to Cytometer > open Detector/Amp, Threshold, Compensation and Status windows.

7- Install unlabeled cells (or any isotype control) on Sample Injecting Port (SIP) to adjust FSC Amp/Gain and SSC voltage in Detector/Amp window.

8- On Cytometer press RUN and MED Flow rate buttons

9- Make sure the Set Up box in Acquisition window is marked, then click on Acquire

10- Change FSC-Amp/Gain and SSC-voltage to put the population of interest on scale (locate the whole visible population in the middle of the dot plot window)

11- Adjust FSC in threshold window to get rid of debris

12- Make a polygon around the population of interest, and take the regions name outside of the polygon region (R1)

13- To save your sample, remove the tube and install DI water

14- Make more dot plots for FL1/FL2 as X/Y axis for two color staining of FITC versus PE (or any other combination that is needed and suits your experiment)

15- Add Quadrant to the plots from tool box (use copy/paste option to add the same Quadrant to other plots)

16- Select all plots except FSC/SSC plot.

17- Go to Inspector window for these plots (multiple objects), and choose Gate > G1=R1 (only the G1=R1 data will be shown in these plots).

18- If desired change the regions color (go to Gate>Gate list>G1=R1, change the color of the checkmark box).

19- Install unlabeled cells and click on Acquire

Note: use Pause & Restart in Acquisition window to refresh the data whenever you make some changes.

20- Adjust FL1 and FL2 Voltage in Detector/Amp window to put the R1 population (unlabeled) in LL quadrant of the FL1/FL2 plot

Note: After these adjustments you may close the Detector/Amp, and threshold windows, because you wont make anymore changes in these settings.

21- Pause & remove the unlabeled tube.

22- To correct the spectral overlap between different detectors use the compensation settings window. These settings are adjusted by using fluorescent positive cells.

Note: For each fluorophore, compensation should be set using the brightest stained population. For example, in a panel containing CD3-FITC, CD4-FITC, and CD8-FITC, set compensation using CD8-FITC, the brightest FITC-stained population in the panel

23- Place FITC (+) control tube on the SIP, and restart Acquisition

24- Adjust FL2 - %FL1 in compensation window to place the FITC positive events in LR quadrant of FL1 versus FL2 plot.

25- Remove the FITC tube, install PE (+) control tube on the SIP, and restart Acquisition.

26- Adjust FL1 - %FL2 in compensation window to place the PE positive events in UL quadrant of the FL1 versus FL2 plot (or adjust FL3 - %FL2 in compensation window to place the PE positive events in LR quadrant of the FL2 versus FL3 plot).

27- Remove the PE tube, install PerCP (+) control tube on the SIP, and restart Acquisition.

28- Adjust FL2- %FL3 in compensation window to place the PerCP positive events in UL quadrant of the FL2 versus FL3 plot (it could be 0.0% change because PerCP does not emit within the range of FL2 detector).

29- Adjust FL4- %FL3 in compensation window to place the PerCP positive events in LR Quadrant of the FL3 versus FL4 plot.

30- Remove the PerCP tube, install APC (+) control tube on SIP, and restart Acquisition.

31- Adjust FL3- %FL4 in compensation window to place the APC positive events in UL Quadrant of the FL3 versus FL4 plot.

32- Close Compensation window.

Note: If you are using two colors, like FITC and PE, you dont need to perform compensation for all the detectors. Only adjust the detectors relevant to your experiment.

33- Pause and Abort in Acquisition window

34- Remove the tube and place DI water, go to Standby mode

35- Save this optimization setting in your folder (Opt-Doc).

(You may also save this setting in the Instrument Setting folder, Cytometer > InstSetting)

Acquisition

At this stage, choose the data saving location and name, specify the data collection information, and then, acquire data.

1- First create an Experiment Storage Folder for this experiment (you may use todays date as its name). For example make a folder in: FACStation hard disk/Home folder/FACS folder/Experiment storage folder (= todays date)

2- Continue with your optimization file that is still open.

3- Click on the Directory/Change button in Acquisition control window

4- Select the Experiment storage folder to save the data files in that folder

5- Click on the File/Change button in the Acquisition Control Window

6- Enter a file prefix name (the prefix can be the date you have performed the experiment), and make sure the file count is set at #1(this refers to your tube #), click OK, (the data file name will be: prefix + tube name)

Optional: You may set the Sample ID/Patient ID as the prefix, in that case you have to enter each samples name in Sample ID box (in Acquisition Window) every time you install a new tube.

7- Go to Acquire>Acquisition & storage, a dialog box will be open

8- Select the setting that fits your experiment, for example: collection criteria, # of events=10000, Gate (G1=R1), Resolution=1024, and etc.

9- Click on OK to exit

10- Go to Acquire>Counters, open Counter window (Zoom on green button to make it big)

11- Deselect Setup checkbox in Acquisition Control Window (this step is necessary to save your data)12- Do a final check (the right tube #, and so on)

13- Install tube #1 on the SIP, Press RUN, wait 5 to stabilize pressure

14- Click on Acquire

15- Live counting status is displayed, wait until you hear the Beep (your 10000 count is done), remove tube 1, install tube 2 (Sample # 2), check if the data file # has changed to #2, and click on Acquire

16- Continue the same procedure for all of your samples.

Note: If one of your samples does not reach the 10000 event counts, you may pause and Save the incomplete data for that sample, and proceed to the next sample. If you Pause and Abort, you have to read that sample again (or the order of your tubes/data files will change).

17- At the end, place a fresh tube containing 1 ml of DI water on the SIPNote: Please perform the clean up protocol (3 ml FACS Clean + 3 ml ddH2O protocol mentioned in Shut Down section), if you are using viscous samples or adhesive dyes such as, Propidium Iodide (PI); Acridine Orange (AO); Thiazole Orange (TO). This will preserve a consistent function of the system.

18- Leave the cytometer on Standby mode, and depressurize the system if the next user is arriving in one hour.

19- Otherwise proceed to shut down section. (For analysis you dont need the cytometer).

Analyzing Data1- Launch CellQuest Pro2- Create a plot (dot plot), and draw a quadrant. You can use the plots in your optimization file for your analysis (OR go to File > BD application>BD CellQuest Pro>Sample file, and double click on Norm001 file)

3- Select a file in:

Inspector window > file > select data files. For example file #1>open (for FCS Data Files you must create a plot first).

4- Select Stat > Quadrant stats, define statistical options in STAT window by using Edit Quadrant Stat>OK

5- Save Data Analysis files as name-analysis file and place it in the same folder as raw data files.

6- Always Log off from your account after you are done

Note: For exporting data you may use a Levy Jenning (LJ) data file format.

Shut down1- Install a tube containing 3 ml of BD FACS Clean Solution (or 10% bleach) with support arm to the side for 1 min (Vacuum ON). Set RUN on HI flow rate mode.2- Move support arm under the tube for 5 min (Vacuum off)3- Repeat steps 1 & 2 with tube containing 3 ml of DI water4- Place a fresh tube containing 1 ml of DI water on the SIP5- Go to Standby mode, and depressurize the system. Then turn off the Power button on the Cytometer6- Log off and Shut down the computerMonthly MaintenanceOnly administrator will perform this procedure.

1- Turn on the cytometer 2- Remove sheath tank and bypass the filter Disconnect the upper tubing of filter from Saline filter port, connect the sheath tube (white) to the port labeled as Saline Filter (Fig 1-4, pg 25)3- Install a spare tank with 1-2 L bleach (10%) or BD FACS clean solution.

4- Install a tube with 3 ml of 10% bleach or BD FACS clean on SIP

5- Press RUN and High flow rate6- RUN for 20-30 min7- Remove tube from SIP8- Repeat step 3-8 using:

a) BD FACS rinse solution,

b) DI water9- Replace original sheath tank10- Connect the sheath filter tubes11- Place a tube with 1 ml of DI water on SIP12- Press standby, depressurize and turn off the cytometer.

Note: If Cytometer doesnt work for three weeks, only use DI water for maintenance to get rid of crystallization. Every 2 months, rinse air filter and dry for 24 hours. Change sheer filter every 4-6 months.

FlurochromeExcitation (nm)EmissionEmitted color

Texas Red488615Red

Phycoerythrin (PE)488575yellow

Fluorescein isothyocianate (FITC)488525Green

PerCP488675Orange

Allophycocyanin (APC)595, 633, 635, 647660Orange

For NERCE related inquiries please contact Christine Anderson, BSL-3 Animal and Tissue Culture Core Laboratory, Rm. 632, [email protected]

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