Splenic immature neutrophil proliferation and mobilization after pneumococcal stimulation. (a) Representative flow cytometry plots (baseline and 24 h) and quantification of Ly6G^hi (blue line) and Ly6G^int (teal line) neutrophil populations at baseline and after S. pneumoniae infection. n = 4–6 pooled from four independent experiments. **, P < 0.01 versus 0 h (baseline). (b) Representative flow cytometry histograms and quantification of Ki67 staining in both Ly6G^hi (blue line) and Ly6G^int (teal line) neutrophil populations at baseline and after S. pneumoniae infection. Positive gates were established by use of an AF488-conjugated isotype control within each experiment. n = 3–4 pooled from two independent experiments. ***, P < 0.001 versus 0 and 12 h. FSC, forward scatter. (c) Time-lapse (0–60 min) spinning-disk confocal images of photoactivated neutrophil cluster mobilization (top left, white box) in the splenic RP at 6 h after saline (gray bar) or S. pneumoniae (blue bar) administration. Green, photoactivated (PA) cells; red, neutrophils; blue, vasculature; multicolored, tracks. Bar, 50 µm. n = 4 pooled from four independent experiments. *, P < 0.05. One-way ANOVA (a and b) and Student’s t test (c) statistical analyses were performed. Data are represented as mean ± SEM.