Fig. 6

Neutrophilic mouse model induced by CFA combined with LPS showed enhanced NETs formation capacity. (A) At 48 h after the last challenge, the percentage of neutrophil populations in CD45(+) leukocytes in mouse peripheral blood and bone marrow were determined by flow cytometry. The representative images in each group are shown. (B) Statistical analysis of the percentage of CD11b(+)Ly6G(+) neutrophils in CD45(+) leukocyte gate of mouse peripheral blood and bone marrow by flow cytometry. (C) Neutrophils were purified from mouse bone marrow and stimulated with PMA (100 nM) or vehicle control for 4 h. Then, neutrophils were stained for myeloperoxidase (MPO, red), citrullinated histone 3 (CitH3, green), and DNA (DAPI, blue) and confocal by immunofluorescence microscope for analysis. Representative images of NETs immunofluorescence. Scale bar = 20 μm. (D) Percentage of NETs area normalized to MPO positive signal in mouse bone marrow neutrophils after PMA stimulation. (E) Representative z-axis images of the NETs immunofluorescence in 0.1LPS + CFA group. Scale bar = 10 μm. (F, G) Western blot analysis the protein expression level of MPO and CitH3 in lung tissue of four groups of mice. Expression is relative to β-Tubulin. Cropped blots are shown, and supplementary Fig. S2 and S5 presents the full-length blots. Data were shown as mean ± SEM; n = 4 or 6. 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