Human PMN purification:

1. To a 15 ml conical tube add

0.2 ml heparin (1000 u/ml)

2 ml 6% dextran T500 (Pharmacia Cat. NO. 17-0320-01) in PBS

10 ml fresh whole blood

Mix well and let sit at RT for 40-60 min, until almost all the erythrocytes have settled into the pellet at the bottom of the tube. The plasma layer contains platelets and leukocytes.

2. To fresh 15 ml tubes add 3 ml of Histopaque-1119, and carefully overly with 3 ml Histopage-1077. Both can be purchased from Sigma.
3. Toa the plasma layer from the dextran sedimentation and layer on top of the step gradient.
4. Centrifuge at 80xg (~700 rpm in a table top centrifuge) at RT for 30 min with the brake off. PMN should be visible as a separate opaque layer between the 1077 and 1119 layers. Mononuclear cells are the layer between medium 1077 and the plasma.
5. Carefully aspirate the PMN from the 1077/1119 interface with a Pasteur pipette. Add to this layer 10 ml HBSS⁼ (ie no ca, mg, or phenol red) containing 4.3% NaHCO₃ and 20 mM HEPES (HBSS). Centrifuge 1500 rpm for 10 min, RT. Wash twice more and finally suspend @ 2x10⁶/ml in HBSS containing 1% HSA. The HSA seems to be important to preserve PMN function; we always use cells within 4h, although it is not clear that this is the longest possible time for storage.