Procedure
1. Pour buffy coat (see Hint #1) from the bag into a 125 ml conical polypropylene centrifuge tube and dilute to 90 ml with sterile endotoxin-free PBS.
2. Add 15 ml of sterile Dextran/PBS and mix by inverting the tube four to five times. If any bubbles appear, they should be removed with a 1 ml plastic pipette (see Hint #2).
3. Leave the mixture undisturbed at room temperature for 20 min to allow the erythrocytes to sediment.
4. Transfer the supernatant with a plastic pipette to a fresh tube and add an equal volume of PBS/EDTA.
5. Centrifuge at 1,000 ×g for 10 min at room temperature to pellet the leukocytes, leaving the platelets in suspension.
6. Pour the supernatant into a container of bleach (see Hint #1), resuspend the cell pellet in 1 to 2 ml of PBS, and transfer the latter to a fresh tube (to avoid contaminating the solution with the platelets that are adhering to the sides of the tube).
7. Bring the cell suspension to a final volume of 50 ml and gently overlay 12.5 ml onto the Ficoll-Hypaque in four 50 ml conical tubes (15 ml Ficoll-Hypaque per tube).
8. Centrifuge at 800×g for 20 min at 12℃ with the BRAKE OFF.
9. The mononuclear leukocytes should appear as a cloudy ring at the PBS/Ficoll interface. Harvest them from the interface with a 2 or 5 ml pipette.
10. Transfer the cells from each interface to a fresh 50 ml tube and fill with PBS.
11. Centrifuge the suspension at 800×g for a minimum of 10 min to pellet the cells. Aspirate the supernatant and resuspend the cell pellets in appropriate buffer (see Hint #3).
Solutions
PBS/EDTA
4.3 mM Sodium Phosphate, Dibasic (Na2HPO4)
pH 7.2
2.7 mM KCl
1.8 mM Potassium Phosphate, Monobasic (KH2PO4)
5 mM EDTA
137 mM NaCl
PBS
4.3 mM Sodium Phosphate, Dibasic (Na2HPO4)
pH 7.2
2.7 mM KCl
1.8 mM Potassium Phosphate, Monobasic (KH2PO4)
Also see Protocol #2152
137 mM NaCl
Ficoll-Hypaque 1077
Incubate 15 ml in four 50 ml tubes
Must be at room temperature
Equilibrate to room temperature
Dextran/PBS 6% (w/v) Dextran
T500 (Pharmacia)
Prepare in sterile PBS (Ca2+, Mg 2+ free)
BioReagents and Chemicals
EDTA
Potassium Phosphate, Monobasic
Ficoll-Hypaque 1077
Dextran T500
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS and institutional policies for proper handling instructions for human blood.
2. The bubbles trap red blood cells.
3. To deplete monocytes, culture the mononuclear cell mixture in several 10 cm dishes for 30 minutes to 1 hour. Monocytes adhere to the plastic dish and the nonadherent lymphocytes can be transferred to a new tube.
