Isolation and NET Formation from Murine Neutrophils

Murine neutrophils were isolated from bone marrow of tibias and femurs as previously described (18 (link), 28 (link)). A BD Aria Plus high-speed sorter was used to sort neutrophils after incubation with PE-anti-mouse-Ly6G and APC-Cy7-anti-mouse-CD11b (BD Bioscience). To generate NETs, neutrophils were then plated to adhere in coated plates for 1 h before 4 h stimulation with phorbol 12-myristate 13-acetate (PMA, 30 nM; Sigma-Aldrich). Neutrophils re-suspended in RPMI were also stimulated as described above for NETs formation in cell culture dishes. After discarding the supernatant, NETs were harvested in 5 ml of new medium and centrifuged at 300 g for 10 min to pellet intact cells. Then, the supernatant was further centrifuged at 20,000 g for 30 min to pellet NETs. Washed NETs were then resuspended in 1 ml of RPMI 1640 + 1% BSA. Nucleosomes and cell-free DNA were measured in washed NET preparations to confirm the presence of NETs (29 (link)).