RSV particles and RSV Fusion protein induce NET formation.

(A) Human neutrophils (2 x 106/mL) were stimulated with RSV (102–104 PFU/mL) or left unstimulated for 3 h at 37°C with 5% CO2. (B) Neutrophils (2 x 106/mL) were stimulated with RSV F protein (0.1–5 μg/mL), PMA (100 nM) or medium alone 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 independent experiments performed in triplicates and represent mean ± SEM. *p5/300 μL) were stimulated with (C) medium, (D) LPS (100 ng/mL), (E) PMA (100 nM) or (F) F protein (1 μg/mL) for 3 h at 37°C with 5% CO2. Cells were then fixed with 4% PFA and stained with Hoechst 33342 (1:2000). Images are representative of at least 4 independent experiments. (G-L) Neutrophils (2 x 105/300 μL) were stimulated with F protein (1 μg/mL) for 3 h at 37°C with 5% CO2. Cells were fixed with 4% PFA and stained with: (G-I) Hoechst 33342 (1:2000), anti-elastase (1:1000), followed by anti-rabbit Cy3 (1:500) antibodies; (J-L) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (1:1000) antibody. Overlay of the fluorescence images are shown in the last panels (I,L). Images are representative of 2 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 50 μm.