Quantifying Neutrophil Extracellular Traps

To prepare NETs, 20 × 10⁶/ml BM isolated neutrophils were plated in 500 µl of serum-free RPMI containing 200 µM Ca⁺ in a 24-well tissue culture plate. Media alone (as control) or A23187 (10 µM) were added, and cells were incubated at 37°C for 5 h. The supernatant was removed and NETs were partially digested in RPMI containing 0.3 U/ml micrococcal nuclease for 10 min at 37°C, followed by centrifugation at 5,000 rpm for 5 min. Supernatants from media alone or supernatants from A23187 treatment were stored −20°C. To disrupt NETs, 40 µl DNase I (10,000 U/ml) (Sigma-Aldrich) was added to 500 µl supernatants from both conditions for 3 h at 37°C, as previously described. NETs were quantified by an ELISA detecting Cit-H3:DNA complexes, as previously described (22 (link)). Briefly, high-binding 96-well ELISA microplates were incubated overnight at 4°C with rabbit anti-citrullinated histone 3 (Abcam, ab 5103) in coating buffer from the Cell Death Detection ELISA kit (cat# 11544675001; Roche). After blocking with 1% BSA (cat# A7906; Sigma) in PBS, samples diluted with 1% BSA in blocking buffer were added, and plates were incubated overnight at 4°C, washed, and anti-DNA-POD (clone MCA-33; Roche) was added for 1 h at room temperature. Following incubation, TMB substrate (cat# T0440; Sigma) was added and absorbance was measured at 450 nm after addition of stop reagent (cat# S5814; Sigma).