How to isolate mononuclear cells from whole blood using density gradient centrifugation

Density gradient centrifugation can be used to isolate mononuclear cells from peripheral blood, cord blood, and bone marrow by exploiting differences in density between the various leukocytes and the density gradient medium. Granulocytes and erythrocytes have a higher density than mononuclear cells and therefore sediment through the density gradient medium layer during centrifugation. To isolate mononuclear cells from peripheral blood, cord blood, and bone marrow, it is recommended to use a medium with a density of 1.077 g/mL, such as Lymphoprep™ or Ficoll-Paque™. This protocol describes how to isolate mononuclear cells (e.g. PBMCs) from whole blood using density gradient centrifugation.

Materials

• Whole blood sample
• Density gradient medium (e.g. Lymphoprep™, Catalog #07801)
• Dulbecco’s Phosphate Buffered Saline with 2% Fetal Bovine Serum (PBS + 2% FBS, Catalog #07905), or other suitable culture medium
• Tube for centrifugation

Protocol

Before You Begin: Ensure all reagents are at room temperature (15 - 25°C).

1. Dilute the blood sample to a 1:1 volume ratio with the appropriate culture medium or PBS + 2% FBS.
2. Add a volume of density gradient medium to a fresh tube according to the specifications of that density gradient medium. If using Lymphoprep™, see Table 1 for recommended volumes and tube sizes.

Table 1. Recommended Volumes and Tube Sizes for Density Gradient Centrifugation using Lymphoprep™