Isolation and Stimulation of Human Neutrophils

Human neutrophils were isolated by centrifuging heparinized venous blood over Histopaque 1119 (Sigma-Aldrich) and subsequently over a discontinuous Percoll (Amersham Biosciences) gradient as previously described (Fuchs et al., 2007 (link)). Experiments were performed in RPMI-1640 (w/o phenol red) supplemented with 10 mM HEPES and 0.05% human serum albumin. Cells were seeded at 10⁵/well (24-well plate) for NET experiments and stimulated with PMA, staurosporine, cycloheximide (Sigma-Aldrich), A23187 (Santa Cruz Biotechnology Inc.), Nigericin (InvivoGen), Candida albicans SC5314 hyphae, GBS, α-hemolysin (generated as previously described [Virreira Winter et al., 2016 (link)]), Staphylococcus aureus (prepared as previously described [Fuchs et al., 2007 (link)]), TNF-α (Thermo Fisher Scientific), z-VAD-FMK (Enzo) or SMAC mimetic (Birinapant, ChemieTek) for 2–6 hr. Where applicable, cells were pre-treated inhibitors for 30 min before stimulation.