To begin, put 15 milliliters of the density gradient medium in a 15-milliliter centrifugation tube and spin it at 1, 000 G for one minute at 20 degrees celsius. Invert the tube containing human blood collected in heparin, and using fire sterilized tweezers, remove the lid from the blood collection tube. Dispense around 10 to 15 milliliters of blood to the density-gradient medium in the tube.
Set the deceleration level of the centrifuge to one and centrifuge the tube with blood at 1, 000 G for 10 minutes at room temperature. Remove the top layer of plasma up to one centimeter above of the buffy coat. Decant the remaining supernatant from the centrifugation tube into another centrifuge tube containing 10 milliliters of PBS.
Reset the deceleration level of the centrifuge to three and centrifuge the tube with PBS and supernatant. Aspirate the supernatant and gently tap the tube to loosen the pellet. Next, incorporate 13 milliliters of isolation medium into the centrifuge tube and wash the inner wall to collect the cells properly.
Using fire sterilized tweezers, position a 100-micrometer cell strainer on the 15-milliliter conical tube. Then filter the cell suspension through the cell strainer into the 15-milliliter conical tube. Dispense equal amounts of cell suspension into two 15-milliliter conical tubes.
Enumerate the cells to calculate the appropriate concentration of isolation buffer. Pellet the cells at 250 G for 10 minutes at four degrees celsius. After removing the supernatant, add the appropriate amount of isolation buffer and pipette around 20 times to loosen the pellet.
