Isolation of Peripheral Blood Mononuclear Cells from Human Buffy Coats

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.