CX3CR1 blockade induces systemic monocyte demargination. (A) 3D-TPLSM image of the skull BM from MacBlue×Cx3cr1^gfp/+ 4 hours after LPS treatment in the presence of F1 (50 µg injected intraperitoneally) (original magnification ×196). Track paths (green line) showing monocyte behavior in the vasculature (red) are represented. (B) Quantification of the relative proportion of crawling and rolling monocytes and their mean velocity in the vascular lumen of the BM sinusoids from MacBlue×Cx3cr1^gfp/+ treated (F1) or not (WT) with CX3CR1 antagonism (bars represent mean ± SEM; data are pooled from different movies out of 2 independent experiments at least. Kruskal-Wallis with Dunn’s multicomparison tests is performed). (C) Quantification by flow cytometry of intravascular Ly6C^high, Ly6C^low monocytes, and neutrophils in WT and treated (F1) after in vivo CD45 intravascular staining (bars represent mean ± SEM from at least 6 different mice per time point out of at least 2 independent experiments; Kruskal-Wallis with Dunn’s multicomparisons test is performed. The significance between mouse strains is indicated by black * for each time point, and Mann-Whitney test is performed). *P < .05; ***P < .001; ****P < .0001; ns, nonsignificant. (D) In vivo 3D-TPLSM image of the peritoneal vasculature showing the defect in ECFP⁺ monocyte adherence to endothelium (red) 4 hours after LPS treatment in the presence of F1 (see supplemental Video 7; original magnification ×150).