This article details the isolation of peripheral blood mononuclear cells (PBMCs) from human buffy coat blood. The process involves using a density gradient medium to separate different cell types based on their densities.
Peripheral blood mononuclear cells, PBMCs, consist of a heterogenous population of specialized immune cells, including lymphocytes, monocytes, and dendritic cells.
To isolate PBMCs, begin with a tube containing a freshly-obtained human buffy coat blood. The buffy coat blood contains PBMCs, along with granulocytes with multilobed nuclei, erythrocytes, and platelets suspended in plasma.
Carefully overlay the buffy coat suspension over an equal volume of a density gradient medium with a desired specific density that is optimal for PBMC separation.
During centrifugation, the different cell types separate into distinct layers, based on the cell densities relative to the gradient medium. Additionally, the polysaccharides in the density gradient medium promote erythrocyte aggregation and sedimentation.
The erythrocytes and granulocytes with higher densities sediment to the bottom of the tube, while the plasma, with the lowest density, forms the topmost layer. The PBMCs, which have lower densities than the density gradient medium, form a cloudy white layer at the plasma-density gradient medium interface.
Remove the top plasma layer. Carefully collect the cloudy layer containing the PBMCs and transfer it to a fresh tube with serum-free medium. Centrifuge to pellet the PBMCs. Discard the supernatant containing platelets and residual gradient medium.
Resuspend the PBMCs in fresh medium and use for further analysis.
To isolate PBMC from human donor buffy coats, use a pipette to slowly overlay 15 millimeters of buffy coat blood, down the side of a 50-milliliter tube, onto 15 milliliters of density gradient medium, and separate the cells by centrifugation. Use a sterile Pasteur pipette to remove the top plasma layer, and carefully transfer the mononuclear cell layer into a new 50-milliliter tube.
Add serum-free RPMI medium to the cells to a final volume of 50 milliliters, and gently invert the tube a few times before centrifuging. Then, resuspend the cells in 20 milliliters of serum-free medium for counting.
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