Method

1. Collect blood with citrate
2. Spin whole blood in 4mL (BD352063) or 15mL tubes,( BD352196) at 2500rpm (1250rcf) at room temperature for 25 minutes
3. Collect buffy coat layer using a pastuer pipette and avoid getting any RBC from below the buffy coat layer( see diagram below)
4. Place the PBL in appropriate sized tube. ( If you start with 1-2mL of blood transfer cells to a 12mL tube(BD352059) and if you start with a larger volume of blood then transfer the cells to a 50mL tube)
5. Wash with medium with 10%FCS
6. Spin the cells down at 1200rpm(300rcf)
7. Discard supernatant by suction with vacuum.
8. Loosen cell pellet
9. Lyse RBC with 2-10mL of 0.1X PBS for less than 20sec
10. Add equal volume 2X PBS and then add 6-20mL of medium with 10% FCS
11. Spin cells down at 1200rpm(300rcf)
12. Wash cells once more with medium with 10%FCS
13. Discard supernatant by suction with vacuum and resuspend cells in appropriate volume
14.  Cells are now ready for use