HumanPeripheralBloodMononuclearCellPreparation

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 .

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 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).

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)