Human neutrophil isolation

Whole blood was obtained from healthy volunteers by phlebotomy into heparinized vacuum phlebotomy vials. RBCs were allowed to sediment for 20 minutes at 20°C after 1:1 mixture with 5% dextran. Supernatant was eluted and centrifuged for 7 minutes at 1000xg at 20°C. Residual RBCs were lysed by resuspending the pellet in ice-cold 0.2% saline for 30 seconds then rapidly bringing solution to isotonicity with 1.6% saline. The cell pellet was resuspended in filter-sterilized 0.9% saline solution equal to starting volume of whole blood and 10mL of Ficoll gradient solution (GE Healthcare BioSciences) was layered at the bottom of a 50mL centrifuge tube. This was then centrifuged for 40 minutes at 1000xg at 4°C. The pellet, now consisting of purified peripheral blood PMNs, was then resuspended in 1mL filter-sterilized phosphate buffered saline (PBS) (pH 7.5 without CaCl₂/MgCl₂) and set on ice. A 1:10 dilution in Trypan blue dye was performed and cells counted; viability of > 95% was confirmed for all experiments.