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Human Peripheral Blood Mononuclear Cell Preparation
This protocol describes a procedure for isolating human peripheral blood mononuclear cells (lymphocytes and monocytes) from a Buffy Coat (obtained from a blood bank). Platelets, red blood cells, and neutrophils are largely depleted from the final cell sample. The isolated cells can be used in chemotaxis assays (see Protocol ID# 2156).
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 X 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 X g for 20 min at 12°C 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 X 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).

PBS/EDTA   4.3 mM Sodium Phosphate, Dibasic (Na2HPO4)
pH 7.2
2.7 mM KCl
1.8 mM Potassium Phosphate, Monobasic (KH2PO4)
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)
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