Human PBMC Isolation

  1. Dilute blood 1:1 with room temperature PBS.
  2. Carefully layer 2 volumes of diluted blood on top of 1 volume of lymphoprep in 50ml falcon tubes (ex: 25ml blood on top of 12.5ml lymphoprep).
    1. Make sure Pipet-Aid is on “medium up” and “slow down”
  3. Place tubes in aerosol containment buckets, and spin at 800xg (~1900rpm) for 20 minutes at room temp. Turn the brake OFF.
  4. Collect the PBMC layer at the interface between plasma and lymphoprep by slowly pipetting up in swirling motion with a glass Pasteur pipette on a pipet-aid.
  5. Collect PBMCs in a 50ml falcon tube. Fill with PBS to wash. Spin at 300xg for 10 minutes, room temp.
  6. Aspirate the supernatant and resuspend the cells in 10mls Buffer 1.
  7. Count cells with 1:10 dilution in trypan blue.
    1. PBMC count: ______________
  8. Spin cells again to wash. Resuspend at 100 million cells/ml