Neutrophil motility in skull BM is increased by G-CSF but not by plerixafor. (a) Snapshots of migrating neutrophils in the BM of LysM-GFP mice during resting state (top left), and 25 min after G-CSF injection (top right). White boxes and circles indicate the region of GFP⁺ neutrophil mobilization from the BM cavity toward the sinusoids (vasculature labeled with TRITC dextran; red). (Bottom) Same images as in the top, with the green signal (neutrophils) omitted. White lines indicate the tracks of migrating neutrophils over the 10-min imaging period. Bars, 40 µm. (b) Mean instantaneous velocity of migrating GFP⁺ neutrophils in skull BM in a LysM-GFP mouse both before and after G-CSF (representative of three independent experiments). (c) The mean migratory velocities (top) and the relative frequency distribution of neutrophil velocities (bottom) at different time points after G-CSF injection (5-min tracking period, n = 3 per group). Each symbol corresponds to an individual tracked neutrophil and the bars indicate the mean velocity during resting, 25 min and 50 min post-G-CSF injection. (d) Snapshots of neutrophils in BM during resting state (left) and after plerixafor treatment (5 mg/kg s.c.; right). White lines indicate the tracks of neutrophils over the 1-h tracking period after plerixafor injection (vasculature labeled with TRITC dextran, red). Bars, 40 µm. (e) Relative GFP fluorescence intensity in the skull BM parenchymal region in LysM-GFP mice treated with G-CSF or plerixafor over time (representative of three independent experiments). (f) Mean instantaneous velocity of migrating monocytes in the BM of CX₃CR1-GFP mouse after G-CSF treatment. Graph shows a representative dataset from three independent experiments. The arrow indicates the time point whereby G-CSF was administered s.c. (g, Top) Mean instantaneous velocity of migrating GFP⁺ neutrophils in skull BM in a LysM-GFP mouse at resting state and after plerixafor treatment (data from three independent experiments). (g, Middle) The mean migratory velocities of neutrophils at different time points after plerixafor injection (30-min tracking period, n = 3 per group). (g, Bottom) The relative frequency distribution of neutrophil velocities (dataset from a representative mouse). Each symbol corresponds to an individual tracked neutrophil and the bars indicate the mean velocity of the whole population. A Student’s two-tailed unpaired t test was performed. Numbers above the frequency distributions indicate the median velocity of the population. PI = post-injection, n.s. = not significant. See also Video 2.