Figure 2

Extracellular superoxide induces NETs via NADPH oxidase. Neutrophils were incubated with media alone (negative control); PMA (100 nM), xanthine oxidase (10 mU/mL) and hypoxanthine (500 nM) to yield superoxide; or glucose oxidase (100 mU/mL) to yield hydrogen peroxide, for 1–8 h at 37°C, in the absence or presence of the NOX inhibitor DPI (10 µM). (A) Western blot of citrullinated Histone H3 levels in neutrophils following treatment as above. β-actin is shown as a loading control. Densitometric quantification charts show mean ± SEM cit-H3 to β-actin, expressed as fold over control. (B) Assay of NET MPO-DNA complexes formed after treatment as above. Shown as mean fold over control. (C) NET formation kinetics in response to superoxide with or without the NOX inhibitor DPI as measured by a fluorescent plate reader assay for extruded DNA fibers (n = 8). NET release is expressed as percentage of total DNA (n = 8). *p < .05 vs control; #p < .05 vs respective uninhibited treatment. All figures represent at least 3 independent experiments for each stimulus.