Figure 2

SSZ induces neutrophil death via NETosis in vivo. (a) WT mice were intraperitoneally injected with 16 mg SSZ or PBS. After 4 or 24 h, the absolute number of CD45.2⁺Ly-6C⁻Ly-6G⁺ neutrophils was determined by counting cells on a microscope using a hemocytometer and analyzing the data using flow cytometry. The average and s.d. of 3 mice are shown. The mean of the number of cells in PBS- and SSZ-injected mice at different time points were compared. NS, not significant between two groups, two-way ANOVA. The data shown are representative of two independent experiments. (b–d) WT mouse were intraperitoneally injected with 1 mg zymosan and 16 mg SSZ or PBS. After 4 h, peritoneal cells were collected. (b) The numbers of total cells (left) and CD45.2⁺Ly-6C⁻Ly-6G⁺ neutrophils (right) were counted as described in (a). (c) Peritoneal cells were stained with 7-AAD, and the proportion of dead cells (7-AAD⁺ cells) was determined using flow cytometry. The values shown are averages and the s.d. of 6 (SSZ (−)) to 7 (SSZ (+)) mice per group (b,c). *P < 0.005, **P < 0.001, Student’s t-test. (d) Peritoneal cells were visualized with anti-citH3 polyclonal antibody as described. Original magnification, ×20. The data shown are representative of three independent experiments.