Transfer the blood collected from the donor to a 50 milliliter tube and add PBS to obtain a final volume of 35 milliliters. Transfer the blood slowly along the wall of the tube containing the polysucrose solution to form a biphasic suspension. Immediately, centrifuge the suspension at 1, 600 G for 30 minutes at 24 degrees Celsius.
After centrifugation, observe the formation of a white ring containing the PBMC's, and collect it by aspirating gently in a 15 milliliter tube. Then, add PBS to the tube containing the PBMCs to obtain a final volume of 10 milliliters. Homogenize the suspension in centrifuge at 1, 600 G for 30 minutes at 24 degrees Celsius.
After centrifugation, wash the cells three times at 1, 600 G for five minutes with 10 milliliters of PBS. Before resuspending the pellet in two milliliters of R10 medium.
