- Dilute the whole blood 1:1 with PBS.
- Layer the diluted sample over a volume of density gradient medium (e.g. Ficoll™, Lymphoprep™) equal to or greater than the initial blood volume.
- Centrifuge for 30 minutes at 600 x g at room temperature, with the brake off. (G to RPM Converter)
- Remove and discard the mononuclear cells which form a layer at the Ficoll:Plasma interface.
- Carefully remove and discard the remaining plasma and density gradient medium.
- Resuspend the red cell pellet in ACK lysis buffer : completely fill the tube with ACK and incubate on ice for 10 minutes.
- Centrifuge for 8 minutes at 300 x g.
- Discard the supernatant and wash the pellet in PBS + 2% FBS.
- Resuspend the resultant polymorphonuclear cells in appropriate medium (e.g. PBS + 2% FBS).
Ficoll™ is a trademark of GE Healthcare Ltd. Lymphoprep™ is a trademark of Nycomed.