Separation of human peripheral blood mononuclear cells

Blood from volunteers (20-60ml) is taken into 50ml Falcon tubes containing 100ul of preservative free heparin (1000 units/ml) and mixed well by inversion.

The blood is diluted 2:1 with RPMI-1640, and 10-25ml of diluted blood is layered onto 5-15ml of Lymphoprep or Ficoll-Hypaque. The gradients are centrifuged with slow acceleration/no brake at 1200rpm for 30 minutes.

The white cell layer at the interface is carefully removed and washed twice in serum free medium. The first wash is centrifuged at 2000rpm for 10 minutes, and the second wash at 1200rpm for 10 minutes.

The cell pellets are resuspended in a small volume of 10% FCS growth medium and counted.