Neutrophil stimulation and NET isolation

Neutrophils were stimulated for 4 hours with 100 nM PMA (Sigma-Aldrich), and NET formation was verified by staining with sytoxgreen (125 nM, Thermo Fisher Scientific). The cell culture medium was aspirated, cells were washed three times with PBS, and NETs were detached by scraping, sonicated, and quantified by PicoGreen assay (Thermo Fisher Scientific). Either DNA or proteins from NETs were isolated by phenol-chloroform extraction, or NETs were digested. The resulting fractions were run on an agarose gel to test the DNA content. To induce degranulation, neutrophils were stimulated with fMLP (10 μM, Sigma-Aldrich).