Standard PBMC Processing Protocol

1. Drain the leukopak into a 250mL conical tube.
2. Spin cells at 500xg for 10 minutes at 20°C, acceleration and deceleration at maximum.
3. Remove supernatant.
4. Resuspend in dPBS + 2% FBS + 1mM EDTA.
5. Layer diluted blood onto 50mL conical tubes containing 15mL LymphoPrep.
6. Spin layered 50mL conical tubes at 800xg for 20 minutes at 20°C, acceleration at 20% of maximum, deceleration at zero.
7. Pipette off and discard top layer.
8. Carefully transfer to white, PBMC interface into new 250mL conical tubes.
9. Add dPBS + 2% FBS + 1mM EDTA to 250mL.
10. Spin cells at 300xg for 10 minutes at 20°C acceleration and deceleration at maximum.
11. Remove supernatant.
12. Repeat steps 9-11 twice for three total washes.
13. Resuspend pellet in Ammonium Chloride Solution.
14. Incubate for 15 minutes on ice.
15. Spin cells at 300xg for 10 minutes at 20°C, acceleration and deceleration at maximum.
16. Resuspend pellet with dPBS + 2% FBS +1mM EDTA and count using acridine orange/ propidium iodide on a Nexcelom Cellometer.
17. Spin cells at 300xg for 10 minutes at 20°C, acceleration and deceleration at maximum.
18. Remove supernatant.
19. Resuspend in appropriate volume of CryoStor CS10 to achieve desired cell density per mL.
** Vials are generated at 15 million, 25 million, 50 million, and 100 million cells. **
20. Aseptically pipette 1.5mL PBMCs into labeled 2.0mL Corning tubes.
21. Place cryovials into an insulated container and place at -80°C overnight for a controlled freeze down.
22. Move cryovials to a liquid nitrogen storage tank for storage until shipment.