Density centrifugation medium, such as Ficoll™ or Lymphoprep™, is widely used for the preparation of mononuclear cell fractions from peripheral blood, cord blood or bone marrow.
- Mix density gradient medium (e.g. Ficoll™, Lymphoprep™) thoroughly before use. Add density gradient medium to an empty tube (see Table 1).
Note: density gradient medium, blood and medium should remain at room temperature for optimum results.
- Dilute the blood 2X with PBS + 2% FBS (see Table 1).
- Layer the blood on top of density gradient medium, being careful to minimize mixing of the blood with the density gradient medium.
- Centrifuge at room temperature at 400 x g for 30 minutes with the brake off. (G to RPM Converter)
- Remove and discard the upper plasma layer.
- Remove and retain the mononuclear cell layer at the plasma-density gradient medium interface.
- Add PBS + 2% FBS and centrifuge at 300 x g for 8 minutes.
- Discard the supernatant and resuspend the resultant mononuclear cells in appropriate medium (e.g. PBS + 2% FBS).
Table 1. Recommended Volumes and Tube Sizes
|Blood (ML)||PBS + 2% FBS (ML)||Density Gradient Medium (ML)||Tube Size (ML)|
Ficoll™ is a trademark of GE Healthcare Ltd. Lymphoprep™ is a trademark of Nycomed.