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. 2016 Sep 19;213(10):1999–2018. doi: 10.1084/jem.20160393

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© 2016 Bajrami et al.

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Figure 4. — MIP-2–induced neutrophil mobilization from the BM is hindered in the presence of G-CSF. (A) GFP⁺ cells in LysM-eGFP mice are positive for Ly6G, a specific marker for neutrophils. Data shown are representative of four experiments with similar results. (B–J) Transgenic mice expressing eGFP under the control of the LysM promoter were i.v. injected with 1 µg MIP-2 or/and 2 µg G-CSF. The mobilization of GFP⁺ cells was monitored in vivo by MP-IVM (also see Videos 1, 2, 3, and 4). (B) Microscopy images at the indicated time points. Representative images from four independent experiments are shown. Arrows indicate neutrophils (time 0) that left upon the treatment. (C) Microscopy images illustrating the migratory patterns of neutrophils (GFP⁺ cells) in the parenchyma and sinusoids of calvarium BM in the first 25 (for MIP-2 alone) or 70 (for G-CSF and G-CSF + MIP-2) min after administration of the indicated cytokines. The tracks of several motile cells are shown. Tracks are sometimes partial because of the depth of the projected image. Bars: (A) 100 µm; (B and C) 50 µm. (D) Tracks of GFP⁺ neutrophils in the BM parenchyma, illustrating the different migrating patterns toward the blood vessels (y axis). GFP⁺ neutrophils were tracked after stimulant administration until they entered the vessels or until the end of the video (25 min for MIP-2 and 70 min for G-CSF and G-CSF + MIP-2). Tracks were obtained from IVM images and are illustrated as starting from a common starting point. (E) Displacement of parenchymal and sinusoidal GFP⁺ neutrophils in the first 25 (for MIP-2 alone) or 70 (for G-CSF and G-CSF + MIP-2) min after administration of the indicated cytokines. Total cell displacement was measured from the position at which each cell started at the beginning of the observation period to its location at the end of the recording. (F) Velocity of parenchymal and sinusoidal GFP⁺ neutrophils. (G) Directionality of neutrophils. (H) Upward directionality. (I) The mean time taken by GFP⁺ neutrophils to migrate from the parenchyma into the blood vessels after the indicated treatments. (J) Percentage of migrating GFP⁺ neutrophils that entered the circulation in the first 60 (for MIP-2 alone) or 70 (for G-CSF and G-CSF + MIP-2) min after administration of the indicated cytokines. Data shown are means ± SD of three experiments (n = 4–6 mice). *, P < 0.01 versus mice treated with MIP-2 alone.