Characterization of mobile and immobilized splenic neutrophils. (a) Representative IVM images, flow cytometry plots, and quantification of splenic neutrophils 24 h after administration of isotype antibody (Ab) or Ly6G (1A8) antibody treatment. Green, neutrophils (neu). mono, monocyte. Bars, 50 µm. n = 5 for isotype and n = 6 for 1A8 antibody pooled from three independent experiments. (b) Representative flow cytometry plots and quantification of Ly6G^hi and Ly6G^int neutrophil origin in the blood, spleen, and bone marrow after parabiosis. n = 4 from two independent experiments. (a and b) Data are represented as mean ± SEM. *, P < 0.01; **, P < 0.01; ***, P < 0.001. Student’s t test (a) and one-way ANOVA (b) statistical analyses were performed. (c) Representative flow cytometry histograms of cell-surface marker expression for both Ly6G^hi (red) and Ly6G^int (blue) neutrophils under steady-state conditions from three independent experiments. (d) Representative immunohistochemistry CD117 staining of the immobilized neutrophil population in the splenic RP from four independent experiments. Bars, 25 µm. (e) Representative nuclear morphology of the immobilized neutrophil population including both banded (white arrowheads) and segmented (blue arrowheads) neutrophils from two independent experiments. Bars, 11.6 µm.