Essential role for NADPH Oxidase-derived ROS on F protein-induced NET generation.

(A,C) Neutrophils (2 x 106/mL) were pretreated with NAC (1 mM) or DPI (10 μM) for 1 h and stimulated with F protein (1 μg/mL) for 3 h at 37°C with 5% CO2. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of 3 separate experiments performed in triplicates and represent mean ± SEM. ***p<0.001 when compared to negative control (NCtrl); #p<0.001 when compared to F protein-treated cells. (B,D) Neutrophils (2 x 106/microtube) were pretreated with NAC (1 mM) or DPI (10 μM) for 1 h, stimulated with F protein (1 μg/mL) for 1 h at 37°C with 5% CO2 and incubated with 0.5 μM CM-H2DCFDA for 30 min. ROS generation was analyzed by flow cytometry using FACSCanto II flow cytometer. Neutrophils gate was based on FSC x SSC distribution. Data are representative of 2 independent experiments performed in triplicates with similar results.