G-CSF treatment does not increase BM vasculature density or permeability. (A) CD31 immunostaining on femoral sections of 129SvJ mice injected with saline for 6 days (Sal) or G-CSF for 4 (G4) or 6 days (G6). Images were captured on a Nanozoomer Digital Pathology C9600-02 scanner (Hamamatsu Photonics) using a 20×/0.7 numeric objective in air and NDP Scan U10074-01 image acquisition software (Hamamatsu). Image analysis was performed in ImageJ software Version 1.41o. (B) Quantification of BM microvascular density showing the number of blood vessels per mm² of BM tissue. Arrows show typical vessels surrounded by CD31⁺ endothelial cells. Data are average ± SD of 4 mice per group. (C) Partition of Evans blue between BM fluid and blood plasma from 129SvJ mice treated with saline for 4 days (Sal), G-CSF for 3 (G3) or 4 (G4) days, or 2 days after a single injection of cyclophosphamide (CY2). Mice were injected retro-orbitally with 2% Evans blue 10 minutes before tissue sampling. Optical density at 620 nm was measured on femoral BM fluids and blood plasma to determine Evans blue concentrations in femoral BM fluids and blood plasma. Volume of blood plasma per femoral BM was then calculated for each individual mouse. Data are average ± SD of 4 mice per group. ***P < .001; *P < .05.