Neutrophil Isolation

Human neutrophils were isolated from the blood of healthy donors age 18–50. All human studies were approved by the University of California, Irvine institutional review board. Whole blood was mixed 1:1 with 3% dextran-PBS for 20 min to sediment red blood cells. The upper layer was overlaid on Ficoll Hypaque (GE Healthcare) and centrifuged at 500 × g for 25 min. The pellet containing granulocytes and RBCs was treated with 1× RBC Lysis Buffer (eBioscience) and spun at 300 × g for 5 min to obtain a granulocyte pellet. Cells were resuspended in RPMI 1640 and counted. Typical purity was >95%. Total bone marrow was isolated from the tibias, and femurs of mice and neutrophils were purified using the EasySep mouse neutrophil enrichment kit (Stemcell), routinely yielding >90% neutrophil population.