CXCR4 blockade inhibits neutrophil trafficking to the BM. (a) Experimental model 2: recipient WT mice were pretreated for 1 h s.c. with PBS, G-CSF, or plerixafor (n = 6 per group) before receiving BM cells from LysM-GFP mice via the cannulated jugular vein. (b) Representative time-lapse images showing neutrophil trafficking to the BM in PBS-treated mice (top), G-CSF–treated mice (middle) or plerixafor-treated mice (bottom). Arrows indicate GFP⁺ neutrophils (green) transmigrated from sinusoids (labeled with TRITC dextran; red) into the marrow space (black region). Bars, 40 µm. See also Video 7. (c, Left) Quantification of GFP⁺ neutrophils in skull BM cavity 2 h after treatment. (c, Right) Number of circulating GFP⁺ neutrophils 2 h after G-CSF or plerixafor treatment (n = 6 per group). Each symbol corresponds to one mouse and the bars represent mean GFP⁺ neutrophil numbers in each treatment group. (d, Left) The experimental procedure involved donor LysM-GFP mice pretreated for 1 h with s.c. PBS or plerixafor before transferring isolated treated BM cells into WT recipient mice via the cannulated jugular vein during imaging. (d, Right) Quantification of PBS-treated and plerixafor-treated GFP⁺ neutrophils in skull BM cavity 2 h after transfer (n = 4–5 per group). All data are shown as mean ± SEM. A Student’s two-tailed unpaired t test was performed. **, P < 0.01; n.s. = not significant. (e) Recipient WT mice were pretreated for 1 h s.c. with PBS or plerixafor before receiving i.v. BM cells from LysM-GFP mice. 1–4 h after transfer of GFP⁺ BM cells, blood and perfused femur were collected and assessed via flow cytometry. (e, Left) The number of GFP⁺ neutrophils detected in the recipient BM. (e, Right) The number of circulating GFP⁺ neutrophils 1–4 h after transfer of BM cells (n = 4 per group per time point). A representative set of data were shown from two independent experiments. All data are shown as mean ± SEM.