Differential effects of G-CSF and plerixafor on neutrophil mobilization in vivo. (a) A schematic representation of the two models established to study neutrophil egress (from BM to blood) and neutrophil ingress (from blood to BM). (b, Top) Experimental model 1: BM cells from LysM-GFP mice were transferred into WT recipient mice at least 4 h before multiphoton imaging of the skull BM. Recipient mice were subsequently injected with PBS, G-CSF, or plerixafor s.c., followed by imaging for up to 2 h (n = 5 per group). (b, Bottom) A representative snapshot of the skull BM 4 h after cell transfer. GFP⁺ cells (green) were predominantly present in the BM cavity outside the sinusoids (labeled with TRITC dextran; red). Bar = 200 µm. (c) Mean instantaneous velocities of transferred GFP⁺ neutrophils in mice treated with G-CSF or plerixafor. Each graph shows a dataset from a representative mouse. (d) Mean GFP⁺ neutrophils egressed from the skull BM after PBS, G-CSF, or plerixafor treatment (n = 5 per treatment group). All data are shown as mean ± SEM. One-way ANOVA analysis was performed. (e) The frequency distribution of neutrophils egressed from the BM into the sinusoidal circulation at various time points after G-CSF or plerixafor injection. Analysis was performed on data pooled from five mice per treatment group. See also Videos 3–5. (f) The number of circulating GFP⁺ neutrophils before and after G-CSF (left) or plerixafor (right) treatment (n = 4 per group per time point). Each line corresponds to a single mouse and shows the number of circulating neutrophils at resting state and 2 h after G-CSF or plerixafor treatment. Student’s two-tailed paired t test was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant.