Murine neutrophils were isolated from bone marrow as previously described [24 (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, Franklin Lakes, NJ, USA). To generate NETs, neutrophils were plated to adhere to coated plates for 1 h before 4 h stimulation with Calcium Ionophore (A23187, 5 µM; Sigma-Aldrich, St. Louis, MO). Neutrophils re-suspended in RPMI were also stimulated as described above for NET 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 with 1% BSA. Nucleosomes and cell-free DNA were measured in washed NET preparations to confirm the presence of NETs [24 (link)].
Free full text: Click here
