• Equipment

  • 10-100 ml Pipette, Eppendorf

  • Rotixa/RP centrifuge, Hettich

  • Biosafety cabinet, Clean Air

  • FACStar, Becton Dickinson

• Disposables

  • Pipettes: 2, 5, 10 ml, Falcon, BD

  • Pipette tips: 1-200 ml, Costar

  • Propylene conial tube: 15 ml, Falcon, BD

  • Polypropylene round-bottom tube with cap: 6 ml (12x75mm), Falcon, BD

  • Bio-freeze vial: 6 ml (12x75mm), Costar.

• Reagents

  • Bovine serum albumine (BSA), Sigma

  • Phosphate buffered Saline (PBS)

  • Wash buffer (PB): PBS/1.0%BSA

  • RPMI-1640 with Hepes, Gibco BRL

  • Fetal bovine serum (FBS), Hyclone

• Antibodies

  • Leu2a-PE (g1;CD8), Becton Dickinson.

  • Leu3a-FITC (g1;CD4), Becton Dickinson.

Methods

• Thaw PBMC gently in 10 ml RPMI with 20% FCS, spin down (4°C, 5', 2000 rpm)

• Resuspend in 2 ml PB, take 40 ml in 20 ml Isoton for cell counting

• Add another 3 mL of PB and spin down (4°C, 5', 2000 rpm)

• In the mean time count the cells with the Coulter Counter

• Resuspend cells in PB at concentration to 10⁷ cells/mL

• Add directly labelled mAb to final concentration of 1:50

• Incubate 30' at 4°C in the dark.

• Wash twice with PB

• Resuspend cells in PB to 3*10⁶/mL in 6 mL tubes. (Max. 4.5 mL)

• Deliver also tubes with 0.5 mL RPMI/20% FCS for harvesting sorted cells

• Sort on FACStar approximately 1-2*10⁶ cells per T-cell subset

• Gate on lymphocyte population in dot plot

• Sort cells with high expression of CD4 or CD8 separately

• Analyze purity of T-cell subsets by analyzing 5,000 events

• Transfer sorted cell suspension to 15 mL conial tube

• Spin cells down (4°C, 5', 3000 rpm)

• Appropriate culture media (i.e. cRPMI for T cell lines, cDMEM for attaching cells) warmed in 37°C waterbath.

• cRPMI = RPMI + 10% FBS+ L-Glut + P/S.

• cDMEM = DMEM + 10% FBS+ L-Glut + P/S + HEPES

• Indo-1 AM, 1 mg/ml in DMSO, store at -20°C, Calbiochem

• Ionomycin, 1 mg/ml in DMSO, store at -20°C, Calbiochem

• Dimethyl sulfoxide (DMSO; Sigma), 10% bleach or ethanol.

• Antibiotics/proteins/growth factors as needed. Monoclonal antibodies must be azide-free. Dialyze against PBS overnight.

• 5 ml FACS tubes, Falcon

Protocol

Indo-1 Loading

1. Count cells and adjust cell concentration to 1 x 10⁷/ml in culture medium.

2. Add 7 µl* of stock Indo-1 per ml of cells and wrap the tube in foil.

3. Incubate the cells with Indo-1 for 45 minutes in a 37°C water bath. Gently mix the loading cells every 10 to 15 minutes to ensure even loading.

4. Wash the cells once with culture medium at 1200 rpm for 8 minutes.

5. Gently resuspend Indo-loaded cells at 5 x 10⁶/ml in culture medium.

6. Store the Indo-loaded cells wrapped in foil at room temperature until the cells can be run on the LSR. Let cells rest for ~15 minutes before starting. Indo-loaded cells should never be placed on ice, because this will inhibit [Ca²⁺]_i signaling.

Sample Running

1. Before starting Indo-1 experiment, turn on circulating waterbath, so it can reach 37°C by the start of your run.

2. Turn LSR on 30 minutes before run. Plug in UV. Make sure the proper filters are in place for running Indo-1/AM: FL5 detector 400BP40, FL4 detector, 510BP20.

3. LSR files for screens, settings and data are found in LSR CAF Stuff user files. Use screen ABW Calcium Flux and settings ABW Calcium. Save data in current week folder.

4. Prewarm 0.8 ml culture medium in several 5 ml FACS tubes to 37°C.

5. Add 0.2 ml Indo-loaded cells (1 x 10⁶)/tube and warm to 37°C for 3 to 5 minutes before running on the LSR.

6. Run cells (400 cells/second) and establish a baseline of [Ca²⁺]_i for 40 to 60 seconds before adding stimulant.

7. FL4-H: M1 gate should contain 80% of the Indo-loaded cells (between 600-800 on X-axis). FL5-H/FL4-H ratio should be just below 200 on the Y-axis.

8. Add 10-50 µl of stimulant and collect for another 5 minutes (this may vary depending on the kinetics of the response).

9. Test ionomycin response first with 5 µl stock ionomycin. This is the maximum stimulation and tests proper loading of the cells (all cells should respond). If indo-loading is not good, the other samples should not be run.

10. After running the ionomycin samples (or other strong stimulants) be sure to flush the lines 1 minute with DMSO or 10% bleach or ethanol, then 1 minute with culture medium to bind any residual ionomycin.

11. Click on pause and save. Print graph.

12. Analysis of data with FlowJo software.

• Indo-1 AM is a fluoescent dye which, as a free ester, absorbs at 349nm. Chelated to calcium it emits at 405nm. It enters cells irreversibly due to cytosolic esterases.

• The optimum concentration of Indo-1 should be determined empirically, but must not exceed 5 mM. At higher concentrations the fraction of Indo-1 that binds Ca2+ will be decreased, resulting in a color change that is less apparent.

• The molecular weight of Indo-1 AM ester is 1009.9 g/M; a 1 mg/ml stock is almost exactly 1 mM.

• Coulter anti-p24 antibody KC57-FITC (cat.no. 6604665)^*. Store at 4°C.

• Pharmingen FITC-conjugated mouse IgG2a, kappa isotype control (cat.no. 20074A). Store at 4°C.

• Falcon 5 ml FACS-tubes (cat.no. 2054).

• BD FACS Lysing Solution (cat.no. 349202)

• BD FACS Permeabilizing Solution (cat.no. 340973)

• PBS/1% BSA: dissolve 4 mg Sigma BSA (cat.no. A-2153) in 400 ml PBS. Filter through 0.22 µm filter. Store at 4°C.

• 2x Storage buffer (2% Formaldehyde, 0.05% Glutaraldehyde): add 5.71 ml 35% Formaldehyde and 200 µl 25% Glutaraldehyde to 94 ml PBS. Store at 4°C.

Protocol

1. For each sample to be analyzed use up to 0.5 x 10⁶ cells (see note).

2. Pellet cells in 5 ml FACS-tube (1100 rpm for 5 minutes).

3. Resuspend in 100 µl of 1x lysing solution.

4. Incubate for 15 minutes at room temperature.

5. Add 4 ml of PBS/1% BSA and spin (1100 rpm 5 min).

6. Remove supernatant and resuspend cell pellet in 100 µl 1x permeabilizing solution.

7. Incubate for 15 minutes at room temperature.

8. Add 4 ml of PBS/1% BSA and spin (1100 rpm for 5 minutes).

9. Remove supernatant and resuspend cell pellet in 100 µl and 1 µl of the appropriate antibody (anti-p24 or isotype control).

10. Vortex at low speed for 1-2 seconds.

11. Incubate for 30 minutes at room temperature (in the dark).

12. Add 4 ml of PBS/1% BSA and spin (1100 rpm for 5 minutes).

13. Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 ml of 1.0% Formaldehyde (1x Storage Buffer) and store them at 2-8°C in the dark. Analyze fixed cells within 24 hours.

Note: for each cell sample that is being tested for p24-expression, be sure to also test control (i.e. test 0.5 x 10⁶ cells with p24-specific antibody and 0.5 x 10⁶ cells with the isotype control antibody) for aspecific sticking.

^*Also available: anti-p24 antibody KC57-RD1 (cat.no. 6604668) for use on GFP-expressing cells. Isotype control: Pharmingen PE-conjugated mouse IgG2a, kappa isotype control (cat.no. 20075A).

• Coulter anti-p24 antibody KC57-FITC (cat.no. 6604665)^*. Store at 4°C.

• Pharmingen FITC-conjugated mouse IgG2a, kappa isotype control (cat.no. 20074A). Store at 4°C.

• Caltag Laboratories Fix Perm permeabilization kit (cat.no. GAS-004).

• Falcon 5 ml FACS-tubes (cat.no. 2054).

• PBS/1%BSA: dissolve 4 mg Sigma BSA (cat.no. A-2153) in 400 ml PBS. Filter through 0.22µm filter. Store at 4°C.

• 2X Storage buffer (2% Formaldehyde, 0.05% Glutaraldehyde): add 5.71 ml 35% Formaldehyde and 200 µl 25% Glutaraldehyde to 94ml PBS. Store at 4°C.

Protocol

1. For each sample to be analyzed use up to 0.5 x 10⁶ cells (see note).

2. Pellet cells in 5 ml FACS-tube (1100 rpm for 5 minutes).

3. Resuspend in 100 µl of reagent A (Fixation Medium).

4. Incubate for 15 minutes at room temperature.

5. Add 5 ml of PBS/1% BSA and spin (1100 rpm for 5 minutes).

6. Remove supernatant and resuspend cell pellet in 100 µl of reagent B (Permeabilization Medium) and 1 µl of the appropriate antibody (anti-p24 or isotype control).

7. Vortex at low speed for 1-2 seconds.

8. Incubate for 15 minutes at room temperature (in the dark).

9. Wash cells with PBS/1% BSA as described above.

10. Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 ml of 1.0% Formaldehyde (1x Storage Buffer) and store them at 2-8°C in the dark. Analyze fixed cells within 24 hours.

Note: for each cell sample that is being tested for p24-expression, be sure to also test control (i.e. test 0.5 x 10⁶ cells with p24-specific antibody and 0.5 x 10⁶ cells with the isotype control antibody) for aspecific sticking.

^*Also available: anti-p24 antibody KC57-RD1 (cat.no. 6604668) for use on GFP-expressing cells. Isotype control: Pharmingen PE-conjugated mouse IgG2a, kappa isotype control (cat.no. 20075A).

• Pharmingen anti-CCR5 antibody 2D7-FITC (cat.no. 36464X)^*. Store at 4°C. Use at 1:5 dilution.

• Pharmingen anti-CXCR4 antibody 12G5-PE (cat.no. 36195X). Store at 4°C. Use at 1:100 dilution.

• Pharmingen FITC-conjugated mouse IgG2a, kappa isotype control (cat.no. 20074A). Store at 4°C. Use at 1:100 dilution.

• Pharmingen PE-conjugated mouse IgG2a, kappa isotype control (cat.no. 20075A). Store at 4°C. Use at 1:100 dilution.

• Falcon 5ml FACS-tubes (cat.no. 2054).

• PBS/1% BSA: dissolve 4mg Sigma BSA (cat.no. A-2153) in 400ml PBS. Filter through 0.22 µm filter. Store at 4°C.

• 2x Storage buffer (2% Formaldehyde, 0.05% Glutaraldehyde): add 5.71 ml 35% Formaldehyde and 200 µl 25% Glutaraldehyde to 94ml PBS. Store at 4°C.

Protocol

1. For each sample to be analyzed use up to 0.5 x 10⁶ cells (see note).

2. Pellet cells in 5 ml FACS-tube (1100 rpm 5 min).

3. Resuspend in 100 µl of cold PBS/1%BSA with appropriate concentration of specific antibody or isotype control.

4. Vortex at low speed for 1-2 seconds.

5. Incubate for 30 minutes in the dark at 4°C.

6. Add 5 ml of cold PBS/1% BSA and spin (1100 rpm 5 min).

7. Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 ml of 1.0% Formaldehyde (1x Storage Buffer) and store them at 2-8°C in the dark. Analyze fixed cells within 24 hours.

Note: for each cell sample that is being tested for chemokine receptor-expression, be sure to also test control (i.e. test 0.5 x 10⁶ cells with chemokine receptor-specific antibody and 0.5 x 10⁶ cells with the isotype control antibody) for aspecific sticking.

^*Also available: anti-CCR5 antibody 2D7-PE (cat.no. 36465X) for use on GFP-expressing cells. Isotype control: Pharmingen PE-conjugated mouse IgG2a, kappa isotype control (cat.no. 20075A).

Date: __________________________

Name: __________________________

Phone: __________________________

Room: __________________________

Principle Investigator: __________________________

Budget Name: __________________________

Budget Number: __________________________

Samples: __________________________

Dyes: __________________________

Time In: __________________________

Time Out: __________________________

Signature: __________________________

When logging your use of the FACSCalibur, include your Principle Investigator’s name, your first name and last initial and your telephone number.

DAILY START UP

1. Turn on the cytometer.
2. Turn on the computer.
3. Fill the sheath tank and verify the following:
   • Tank is filled (~3L).
   • Cap is tight.
   • Tank is pressurized.
   • Tubing is not kinked.
4. Empty the waste tank and verify the following:
   • Tank contains 400 mL bleach.
   • Tubing is not kinked.
5. Purge bubbles from sheath filter.
6. Allow the cytometer to warm up for 5 minutes.

DAILY CALIBRATION (FACSCOMP)

1. Label one 12 x 75 mm tube A and another B.
2. Add 1 mL of sheath to tube A. Add 3 mL to tube B.
3. Add one free-falling, complete drop of the appropriate CaliBRITE beads to each tube:
   • Unlabeled and APC beads to tube A.
   • Unlabeled, FITC, PE, PerCP and APC beads to tube B.
4. Empty the waste tank and verify the following:
   • Tank contains 400 mL bleach.
   • Tubing is not kinked.
5. Cap the tubes and mix by gentle inversion.
6. Select FACSComp from the Apple Menu.
7. Fill in the Operator field. Click Accept.
8. The set up information window appears. Unless a new lot of CaliBRITE beads is used for the first time, this information does not need to be changed. Click Run.
9. Make sure the sample flow rate is set to HI and the cytometer is set to RUN. Install tube A.
10. Click Start to begin PMT adjustment and time-delay calibration.
11. Remove tube A and install tube B.
12. Click Start to begin compensation adjustment and sensitivity tests.
13. Remove tube B, install distilled water tube and set cytometer to STANDBY.
14. Click Quit to quit FACSComp. The report will be printed automatically. Please put it in the appropriate section of the blue FACSCalibur folder.

SAVING DATA

1. When designating your data file name, place your initials and the date (ABCyymmdd) in the custom prefix box. Assign a file count number and this will be appended to the date and will increment as you run each sample.
2. The designation folder should be Flowdata. You may create a folder within Flowdata with your name.
3. At the end of your run, transfer your data files to a Zip disk or your directory on Ireland.

DAILY SHUTDOWN

1. Install a 12x75mm tube containing 3 mL of 10% bleach on the sample injection port (SIP).
2. Leave the support arm to the side for 1 minute.
3. Place the support arm under the tube, make sure the cytometer is in RUN, and let it run at HI flow rate for 5 minutes.
4. Repeat steps 1 through 3 using distilled water.
5. Leave a tube containing 1 mL of distilled water on the SIP with the support arm under the tube.
6. Set the cytometer to STANDBY mode.
7. Shut down the computer.
8. Turn off the cytometer.

PROBLEMS

Record any problems in the Problem Log or let Angélique (2-6077) or Sherry (2-6067) know directly.

Flowdata user screens, settings, and data SCREEN_displays select a screen display.

CONNECT TO CYTOMETER

Acquire connect to cytometer. The Acquisition Control Dialog Box with acquisition control functions: Setup, Acquire, Restart, Save, Abort appears. The setup box should have an X mark until you are ready to start saving data. Move the window to the side.

The counter window is also under Acquire.

RETRIEVE INSTRUMENT SETTINGS

Cytometer Instrument Settings OPEN user screens, settings, and data settings double click on a file SET (wait for 5 seconds, while the world turns) DONE.

TO SAVE DATA FILES

ASSIGN DATAFILE NAME AND COUNT

Acquire Parameter Description Folder (Flowdata/user screens, settings, and data/FlowDataFiles) File (custom prefix = initials+YYMMDD, and the file count = 1) Enter sample information in the comment box, if you will be using ReproMan or ReproMac for analysis. Enter sample information in the Sample ID box, if you will be using CellQuest for analysis. Information can be entered in both places.

ASSIGN STORAGE INFORMATION

Acquire Acquisition and Storage Acquisition Gate (Accept all), Collect criteria (Event count = # of cells to be counted, total or in a gate). Storage gate (All or gated) If a gate is selected, events outside the gate will NOT be saved for future analysis. Resolution (256 or 1024) Parameters saved (turn off the parameters not in use) OK.

TO CHECK SAMPLE SETTINGS

The Setup box should have an X, while you run your negative background etc. to adjust the high voltages and compensations. Adjustments can be made either on the cytometer or in CellQuest. To make the adjustments in CellQuest, select Cytometer Detectors/Amps. Select Threshold and Compensation, if these will be adjusted.

RUN SAMPLES

Turn the knob from Standby to Run. Swing the arm to the side. Remove sheath tube. Put on your sample tube. Swing the arm to the left. Pulse lights should appear if cells are flowing. Select Acquire in the acquisition control box. Make adjustments as necessary. When putting the next tube on, there is no need to switch to Standby. The sample probe is continually flushing while in the run mode. When satisfied with the settings, they must be saved, if you want to use them again.

TO SAVE NEW INSTRUMENT SETTINGS

Cytometer Instrument Settings SAVE Find settings folder within user screens, settings and data folder type in appropriate name SAVE DONE.

TO ACQUIRE DATA

Acquisition Control Box Pause Abort remove the X from the Setup box. When the correct sample is flowing, select Acquire. A tone will sound when collection is finished and the file count will increment. Change the comment or sample ID info and run the next sample.

TO QUIT CELLQUEST

File Quit SAVE if you wish to keep screen changes made during this run. The settings are NOT saved as part of the screen. They must be saved separately as explained above.

TRANSFER DATA FILES

Copy all your data to your directory on Ireland or on ZIP disks. Do NOT save your data on the FACS computer only. ALL files in the temporary directories will be erased weekly on Monday morning.

PLEASE RECORD ANY CELLQUEST OR INSTRUMENT PROBLEMS ON THE CLIPBOARD LOCATED ON TOP OF THE FACSCALIBUR. THANK YOU!

FURTHER READING

There is excellent FACS documentation at the website of the Cell Analysis Facility of the Department of Immunology: http://nucleus.immunol.washington.edu/Research_facilities/cell_analysis.html