Fig. 3.

Abundant NETs occur in neutrophilic asthma models. (A) Neutrophil expression in CD45(+) leucocytes in mouse peripheral blood and bone marrow were determined by flow cytometry within 48 h after the last challenge. The representative images in each group are shown. (B) Quantification: the percentage of CD11b(+)Ly6G(+) neutrophils in the CD45(+) leucocytes gate of mouse peripheral blood and bone marrow by flow cytometry. (C) In vitro NET-formation assays with purified neutrophils of mouse bone marrow neutrophils stimulated with PMA (100 nM) or RPMI 1640 for 4 h. Then the neutrophils were stained PI and analyzed by immunofluorescence confocal microscopy. Representative immunofluorescence images of NETs, Scale bar = 20 μm. (D) Comparison of the percentage of NETosis cells in each group upon PMA stimulation and control (Ctrl) stimulation. (E) After stimulated with PMA, the neutrophils were stained for myeloperoxidase (MPO, red), citrullinated histone 3 (CitH3, green) and DAPI (nuclear staining, blue) and analyzed by immunofluorescence confocal microscopy. Representative immunofluorescence images of NETs, Scale bar = 20 μm. (F) Percentage of NETs area normalized to MPO positive signal in mouse bone marrow neutrophils after PMA stimulation. (G, H) Western blot was used to detect the levels of MPO and CitH3 protein in lung tissue of five groups of mice. Expression is relative to β-Tubulin. Cropped blots are shown, and supplementary Fig. S1 and S4 presents the full-length blots. Data were shown as mean ± SEM; n = 4. Significance between groups was calculated using one-way ANOVA with Tukey’s post hoc method. *p < 0.05, **p < 0.01 and ***p < 0.001