Quantification and Visualization of NET DNA Release

(a) Quantitation of DNA release: Neutrophils (5 × 10⁵/500 µL media containing 2% (v/v) FBS) were seeded into wells of a 24-well culture and incubated for 1 h at 37 °C. APPA (100 µg/mL) was then added and incubated for 10 min before stimulation with 100nM PMA for 3 h at 37 °C. After incubation, NET DNA was isolated using Micrococcal nuclease (500 mU, Sigma) and quantified utilizing picogreen (Promega) and a DNA calibration curve. (b) microscopic visualisation: neutrophils were seeded and incubated as described above. Following incubation cells were fixed on cover slips, stained with neutrophil elastase antibody and DAPI (Thermo-Fisher) before being viewed microscopically on a Leica TCS SPE (Papayannopoulos et al. 2010 (link)).