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|  | | | | 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)
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)
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