Fig 2. Effect of different treatments on F protein-induced NETs generation.

Human neutrophils (2 x 10⁶/mL) were stimulated with: (A) F protein (1 μg/mL) or LPS (100 ng/mL) in the presence or absence of DNase-1 (100U/mL); (B) F protein (1 μg/mL) or LPS (100 ng/mL) in the presence or absence of polymyxin B (Pmx B, 1 μg/mL); (C) F protein (1 μg/mL), boiled F protein (1 μg/mL, 10 min at 100°C) or F protein (1 μg/mL) treated with proteinase K (1 mg/mL for 90 min) for 3 h at 37°C with 5% CO₂. (D) F protein solution was treated with monoclonal anti-F protein (10 μg/mL) or isotype-matched (10 μg/mL) antibody and neutrophils (2 x 10⁶/mL) were stimulated with these preparations 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 at least 2 independent experiments performed in triplicates and represent mean ± SEM. *p<0.05; ***p<0.001 when compared to negative control (NCtrl); #p<0.05 when compared to LPS- or F protein-treated cells.