Reagents:

• AIS MicroCellector anti-CD8 T-25 flasks (AIS, 650139E)

• Dulbecco’s Phosphate Buffered Saline/Calcium Magnesium Free (D-PBS/CMF)

• Ethylenediaminetetraacetic Acid (EDTA)

• Human Gamma Globulins, Cohn fraction II, III (Sigma G4386)

• cIMDM (10% FBS, Pen/Strep)

Solutions:

• D-PBSE/CMF: D-PBS/CMF containing 1mM EDTA

• D-PBSE/CMF/0.5% Ig: dilute human gamma globulins to 0.5% in D-PBS/CMF, heat at 56°C for 30 minutes. Cool to 2-10°C, add EDTA to 1mM final concentration, filter sterilize over 0.2 µm filter.

Protocol:

1. Add 10 ml D-PBSE/CMF to AIS MicroCELLector T-25. Swirl.

2. Incubate for 1 hour at room temperature.

3. Shake vigorously for 30 seconds and aspirate D-PBSE/CMF.

4. Add 10 ml D-PBSE/CMF and shake flask again. Remove D-PBSE/CMF. Repeat 2 additional times.

5. Resuspend PBMC in 4 ml P-DBSE/CMF/0.5% Ig per 20 x 10⁶ cells.

6. Incubate for at least 15 minutes at room temperature.

7. Just before adding the PBMC to flask completely aspirate remaining D-PBSE/CMF from flask.

8. Slowly add 20 x 10⁶ PBMC in 4 ml per flask.

9. Incubate for 1 hour at room temperature (binding of CD8⁺ cells).

10. Gently rock flask from side to side.

11. Collect non-adherent CD8^- cells. Gently wash flask with 4 ml of D-PBSE/CMF. Repeat 3-5 times. Combine all CD8^- cells.

12. Spin at 1500 rpm for 10 minutes, resuspend pellet in cIMDM, count and cryopreserve.

13. Add 5-10 ml cIMDM to the flask.

14. Incubate for 48-72 hours at 37 °C in 5% CO2.

15. Collect CD8⁺ cells. Wash flask with 5-10 ml cIMDM. Combine all CD8⁺ cells.

16. Spin at 1500 rpm for 10 minutes, resuspend pellet in cIMDM, count and cryopreserve

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.