Density gradient centrifugation is the most common method for isolating peripheral blood mononuclear cells (PBMCs), namely lymphocytes and monocytes. For this isolation procedure, PBMCs are centrifuged in the presence of density gradient media, such as Ficoll or Ficoll-Paque medium. Each cell population exhibits a unique migration pattern through the medium (related to the density of the cell), creating distinct layers that each contain specific cell populations. The cells can then be isolated by extracting the respective layer.
Instructions for Isolating PBMCs from Whole Blood Samples
For PBMC isolation via density gradient centrifugation, anticoagulant-treated or defibrinated blood specimens are carefully layered on top of Ficoll medium in a tube. The tube is then briefly centrifuged, causing the cells to separate into several layers. Erythrocytes (red blood cells) aggregate together, and these dense cell clusters sediment on the very bottom of the tube. Granulocytes are located in the Ficoll medium layer, directly above the erythrocytes. The top visible layer is plasma. The plasma/Ficoll medium interface contains both PBMCs and platelets. To isolate PBMCs, this interface is carefully removed, washed with salt-buffered solution, and then centrifuged. The supernatant, containing platelets, Ficoll, and plasma, is removed, leaving a pellet of purified PBMCs.
The instructions below are based on a GE protocol, and this video also provides step-by-step instructions for PBMC isolation via density gradient centrifugation.
Dilute the blood sample in balanced salt buffer (e.g., phosphate-buffered saline) at a 1:1 (volume:volume) ratio. For example, dilute 2 mL of blood in 2 mL of buffer. Gently mix with a Pasteur pipette.
Thoroughly mix the Ficoll-Paque medium by repeatedly inverting the stock bottle; then, add the medium to a clean, new centrifuge tube.
Layer the diluted blood sample on top of the Ficoll-Paque medium, carefully ensuring that the blood and medium do not mix.
Centrifuge the tube at room temperature (i.e. 15-25°C) for 30 minutes at 400 g with the brake off/soft stop.
Remove the tube from the centrifuge, noting the visible layers. The top layer contains plasma, the middle layer is composed of Ficoll medium and granulocytes, and the bottom layer comprises erythrocytes. The PBMCs are located between the top plasma layer and the Ficoll medium.
Two techniques can be used to isolate the PBMCs at the plasma/Ficoll interface.
a. Use a clean pipette to carefully remove and discard (or save for later use) the upper plasma layer without disturbing the PBMC-containing plasma/Ficoll interface. Then, transfer the PBMCs to a new, clean tube.
b. Insert a clean pipette through the plasma layer and remove the interface layer containing PMBCs. Avoid extracting plasma or medium, which will contaminate the PBMCs. Gently transfer the PBMC layer to a clean, new tube.
Estimate the interface volume, add a 3x volume of balanced salt solution, and gently suspend the PBMCs (e.g., for a 1-mL interface, add 3 mL of PBS).
Centrifuge the PBMCs at 200 g for 10 minutes at room temperature and remove the resulting supernatant, which contains any contaminating Ficoll medium or platelets/plasma proteins.
Repeat steps 7-8 once more to maximize sample purity.
Research Applications for Purified PBMCs
Once purified, PBMCs can be used in countless assays to provide valuable insights into health and disease, and individual PBMC subpopulations can be further isolated for more specific studies. For example, one PBMC subpopulation, CD8+ T cells, has revolutionized immuno-oncology treatment. CD8+ T cells are removed from patients with cancer, genetically modified, and then reinfused into the patient. In the patient, these cells activate the patient's immune system to target and eradicate cancer cells. Other PBMC subpopulations, such as natural killer cells, also have promising potential therapeutic applications.
Revised on 6/22/18 by Rachel Lane PhD, RD