1. Label 15 ml centrifuge tubes per sample and add 8 ml of R10 complete medium.
2. Set water bath at 37°C.
3. Retrieve cryovials of required sample from Liquid nitrogen and place directly on dry ice.
4. Thaw no more than 2 cryovials at a time. Place cryovials in a 37°C water bath until suspension is reduced to pea-size pellet.
5. Dry off outside and wipe with 70% ethanol.
6. Add 1 ml of cell culture media to the thawed cells dropwise.
7. Transfer cell suspension to the 15 ml tube.
8. Centrifuge at 1000 rpm for 10 minutes.
9. Aspirate supernatant and resuspend pellet in 5 ml R10 complete medium.
10. Count viable cells in Neubaer chamber using 1:4 dilution (25 µl cells, 75 µl trypan blue.)
11. Resuspend at a final concentration of 1-2 x 10⁶ live PBMCs/ml in R-10 media.
12. Incubate at 37°C for 12-18 hours (overnight).

1. Concentrate cells to between 0.5-1 x 10⁶ cells per 200 ul R-10 media after washing the above-prepared cells and dispense in appropriate labeled wells of 96-well plate.
2. Add CD28/CD49d costimulatory antibodies (2 ul per well i.e. 1 ug/ml) to all wells.
3. Add peptide (at final concentration of 2 ug/ml) and SEB (1ug/ml final concentration) to appropriate labeled wells. Do not add anything to the unstimulated control wells.
4. Mix well and incubate covered for 2 hours at 37 C.
5. Add BFA (4 ul per well i.e. 10ug/ml concentration) to all wells.
6. Mix well and incubate covered for 4 hours at 37 C.

Cells may be stored at 4-8 °C for up to 16-18 hours.

7. Add 30 µl of PBS-EDTA to each well and mix well. Incubate for 15 minutes at room temperature.
8. Centrifuge plate at 1200 rpm for five minutes.
9. Aspirate supernatant (by flicking the plate carefully) and resuspend each well with 100 µl of 1X BD FACS Lysing Solution. Incubate at room temp for 10 minutes.

At this point the plate may be wrapped in foil and placed in –70°C freezer.

10. Add 100 µl of Wash Buffer to each well. Centrifuge at 1500 rpm for five minutes.
11. Aspirate supernatant and resuspend cells with 200 µl of 1X BD FACS Perm II per well.
12. Incubate for 10 minutes Centrifuge at room temperature at 1500 rpm for five minutes.
13. Aspirate supernatant and resuspend cells with 200 µl of Wash Buffer per well.
14. Add BD antibody panels and above stimulated cells to the appropriate labeled FACS tubes.
15. Mix well and incubate at room temperature for 30-60 minutes.
16. Add 1ml Wash Buffer per well. Centrifuge 1500 rpm for 10 minutes.
17. Aspirate supernatant and resuspend pellet with 225 µl 1x PBS.
18. Keep in dark until acquisition, which should be performed within 2-18 hours.

• BFA
• Add 1ml DMSO directly to a 5 mg vial of Brefeldin A. Cap the vial and shake to dissolve all the powder. Freeze aliquots of 20 µl each at –20 C. These are thawed on day of use and diluted 1:10 by adding 180 µl sterile PBS. The diluted stock may be stored at 4 C for up to one week

• EDTA
• 20 mM EDTA in PBS: Add 744 mg EDTA to 100 ml 1x PBS. Adjust the pH to 7.2-7.4 with 1 N NaOH. Filter sterilize and store at 4 C.


• 1X BD FACS Lyse
• Dilute 10X stock with deionized water. Store at room temperature.


• 1x BD FACS Perm 2
• Dilute 10X stock with deionized water. Store at room temperature.


• Wash Buffer
• Add 2.5 g BSA to 50 ml 1X PBS. Heat in water bath to facilitate dissolving. Remove 55 ml of PBS from a new bottle of 1X PBS. Add 5% BSA solution and add 5 ml 10% NaN3 to bottle. Filter sterilize. Store at 4 C.


• R-10 Complete media
• o Add 55 ml heat inactivated FBS to 500 ml bottle of RPMI-1640 medium. Add 5 ml aliquots of Penstrep and L-Glutamine.

Dr. Madhu Vajpayee
Associate Professor
HIV &Immunology Division Department of Microbiology,
All India Institute of Medical Sciences,
New Delhi India 110029.