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 #18060)
• 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™
   
   
   Blood (mL)

   PBS + 2% FBS (mL)

   Lymphoprep™ (mL)

   Tube Size (mL)

   Blood (mL)

   1

   PBS + 2% FBS (mL)

   1

   Lymphoprep™ (mL)

   1.5

   Tube Size (mL)

   5

   Blood (mL)

   2

   PBS + 2% FBS (mL)

   2

   Lymphoprep™ (mL)

   3

   Tube Size (mL)

   14

   Blood (mL)

   3

   PBS + 2% FBS (mL)

   3

   Lymphoprep™ (mL)

   3

   Tube Size (mL)

   14

   Blood (mL)

   4

   PBS + 2% FBS (mL)

   4

   Lymphoprep™ (mL)

   4

   Tube Size (mL)

   14

   Blood (mL)

   5

   PBS + 2% FBS (mL)

   5

   Lymphoprep™ (mL)

   10

   Tube Size (mL)

   50

   Blood (mL)

   10

   PBS + 2% FBS (mL)

   10

   Lymphoprep™ (mL)

   15

   Tube Size (mL)

   50

   Blood (mL)

   15

   PBS + 2% FBS (mL)

   15

   Lymphoprep™ (mL)

   15

   Tube Size (mL)

   50
   
   
3. Gently layer the diluted blood on top of the density gradient medium. Take care not to mix the two layers.
4. Centrifuge at 800 x g for 20 - 30 minutes with the brake OFF.
5. Carefully harvest the cells by inserting the pipette directly through the upper plasma layer to the mononuclear cells at the interface. Alternatively, you can first remove the upper layer and then collect the cells.
6. Wash the harvested cells twice in the appropriate buffer. Cells are now ready for downstream applications.