Immunofluorescent Analysis of NETs

NETs were analyzed using immunofluorescent staining as reported previously (Ullah et al., 2017 (link)). Neutrophils were seeded on 8-mm sterile glass coverslips at a density of 2 × 10⁵ cells/well with RPMI 1640 containing 10% FCS for 2 h and then were infected with D39 (MOI = 1) for 4 h. After infection, cells were fixed with 4% paraformaldehyde for 30 min. Then, cells were washed with PBS three times, and permeabilized with 0.1% Triton-X 100. After blocking with 5% BSA, cells were incubated with rabbit monoclonal anti-Histone H3 (citrulline R17; Abcam, Cambridge, United Kingdom) for 2 h and goat polyclonal secondary anti-rabbit IgG-H&L Alexa Fluor^® 594 (Abcam, Cambridge, United Kingdom) for 1 h. Finally, cells were mounted using Mounting Medium (Solarbio, Beijing, China). DAPI was used to stain cell nuclei. Slides were observed on a fluorescent-inverted microscope (Olympus, Japan).