Isolation of Human Neutrophils from Buffy Coats

Human neutrophils were isolated from buffy coats as previously described (57 (link)). In brief, dextran T 500 (3%) (Carl Roth GmbH, Germany) (3% dextran T 500 in 0.9% NaCl, at 4:1) was added to the buffy coats in 15-mL tubes, and erythrocytes were sedimented at gravity for 20 min. The white blood cell layer was removed carefully, and cells were pelleted by centrifugation at 200 × g for 20 min. The cell pellet was resuspended in 12 mL of Hanks’ balanced salt solution (HBSS; Lonza Cologne GmbH, Germany), layered on top of 3 mL Ficoll-plaque Plus (GE Healthcare, Germany), and centrifuged at 400 × g for 20 min. Next, residual erythrocytes were lysed in lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, and 10 mM EDTA [pH 7.4]). The neutrophils were washed (200 × g for 10 min), suspended in HBSS, and used immediately.