Isolation of peripheral blood mononuclear cells..

1. Obtain blood sterile into heparinized vacutainer tubes (green top). Count on 1-2x106 lymphocytes per mL of whole blood 2. Open tubes in the laminar flow hood, and pipet blood from each glass tube into a 50 mL tube. 3. Pipet 10 mL of PBS into each vacutainer tube, and transfer to the same 50 mL tube (i.e. rinse vacutainer tubes). 4. Now you have ~ 1:1 dilution of blood with PBS. 5. Underlay this carefully with 10 mL of FICOLL. 6. Spin at 1600 rpm for 30 minutes, brake off (since you have a gradient i.e. cells are in layers). 7. Resultant layers are approximeately from top to bottom: Plasma platelets -- PBMC Ficoll red blood cells (with granulocytes). 8. Pipette off most of plasma, and discard. 9. Carefully aspirate the buffy coat with PBMCs from 3 large (50 mL) tubes and transfer to one new 50 mL tube. Including some plasma is no problem, but you should try to minimize aspirating Ficoll. 10. Discard the remainder of the Ficoll and red blood cells in closed tubes. 11. Add enough PBS to the PBMCs to make up 50 mL. Spin at 1200 rpm for 10 minutes, brake on (cell pelleting). 12. Decant the supernatant, loosen pellet, and wash once again in PBS. 13. Decant supernatant, loosen pellet, and add ~30 mL of PBS. Mix tube well. 14. Remove 10 :l of well-mixed cell suspension into a well of 96 well plate. Add 10 :l of Trypan blue. Mix well, and load a hemacytometer chamber. 15. Under the microscope count the number of cells in the central 25 squares. Multiply by 2 (dilution factor with trypan blue) and by 104 = cell number/ml.

For example: 128 cells x 2 x 104 =256x104 = 2.56x106/ml x 30 ml of PBS = 76.8x106 PBMC total D.Bienzle, 1.08.2003v