F protein-induced NET formation is dependent on TLR-4 activation.

(A) Human neutrophils (2 x 106/mL) were pretreated with monoclonal anti-TLR4 (10 μg/mL) or isotype-matched (10 μg/mL) antibody for 1 h and stimulated with F protein (1 μg/mL) or medium 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 3 separate experiments performed in triplicates and represent mean ± SEM. *p<0.001 when compared to negative control (NCtrl); #p<0.05 when compared to F protein-treated cells. (B) Neutrophils (2 x 105/300 μL) were pretreated with anti-TLR4 (10 μg/mL) for 1 h at 37°C with 5% CO2 and stimulated with F protein (1 μg/mL) or medium for 3 h at 37°C with 5% CO2. Cells were fixed with 4% PFA and stained with Hoechst 33342 (1:2000). Confocal images were taken in a Zeiss LSM 5 Exciter microscope. Image is representative of 2 independent experiments. Scale bars = 50 μm.