CCR2 and CX3CR1 control BM parenchymal and vascular monocyte release following intraperitoneal LPS injection. Kinetics of Ly6C^high (red), Ly6C^low monocytes (blue), and Ly6G⁺ neutrophils (green) mobilization in the BM (A) and the blood (B) after intraperitoneal injection of 100 ng/kg LPS in MacBlue×Cx3cr1^gfp/+ mice. Dot plots are gated on CD11b⁺NK1.1^-−Ly6G⁻ cells (percent ± SD of the gated populations are indicated). (C) Graph shows the quantification of BM CD45⁺ monocytes and neutrophils after intravascular staining (for all graphs, data represent mean ± SEM of absolute numbers of each subset per thighbone or per milliliter of blood quantified by flow cytometry [n = 6-12 mice for each time point out of 3 independent experiments]; Kruskal-Wallis with Dunn’s multicomparisons test is performed. Only the significance for each time point compared with untreated mice [t0] is indicated). (D) Representative 3D-TPLSM images of the skull BM from MacBlue×Cx3cr1^gfp/+, MacBlue×Cx3cr1^gfp/+Ccr2^−/−, and MacBlue×Cx3cr1^gfp/gfp mice at different time points before and after LPS injection (original magnification ×115). Track paths of vascular ECFP⁺ cells are represented in green. BM sinusoids are labeled by Rhodamin-Dextran (red); bone matrix is detected by SHG (blue), and monocytes are in white/cyan. (E) Flow cytometry quantification of BM CD45⁺ monocytes and neutrophils after intravascular staining in WT, CCR2-, and CX3CR1-deficient mice bars represent mean ± SEM from 6-12 different mice per time point out of at least 3 independent experiments. Kruskal-Wallis with Dunn’s multicomparisons test is performed. The significance for each time point compared with untreated mice [t0] is indicated by colored *. Significance compared to 1 h is indicated by colored $$ (P < .01). Significance between mouse strains is indicated by black * for each time point; Mann-Whitney test is performed. *P < .05; **P < .01; ***P < .001; ****P < .0001). (F) Quantification of the relative proportion in the number of crawling and rolling monocytes in BM sinusoids. Bars represent mean ± SEM calculated from 2 to 5 different mice per group. (G) Mean velocity of ECFP⁺ vascular cells in the BM upon LPS injection (data are pooled from at least 3 different mice per group and per time point. One-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison tests is performed).