Analysis of the hypoxic status of BM and HSCs. (A) In vivo HO perfusion efficiency analyzed simultaneously in the thymus, spleen, and BM. Negative cells were defined using a noninjected control, based on both HO blue and HO red fluorescence (see inset: fluorescence-activated cell sorter [FACS] analysis; upper panel, control; lower panel, HO injected). The gates indicated in the FACS panels (inset) were used to analyze the percentage of HO-negative cells from animals infused with HO, 5 and 10 minutes before being euthanized. Average values are displayed ± standard deviation (SD) (n = 3). *P < .05; **P < .005. (B) Frequency of PIM-positive leukocytes within the designated compartments. Average ± SD is displayed (n = 3). Percentage (±SD) of HO-negative cells (C) and PIM+ cells (D) within the LK, LSK, and LSK-CD150 cells from Dia, Epi, and Cal (n = 3 and 3, respectively). Specific HO (E) and PIM (F) uptake within HSPCs from Dia, Epi, and Cal (n = 7 from 2 independent experiments and n = 3, respectively). *P < .05 or **P < .005. Cal, calvaria; Dia, diaphyses; Epi, epiphyses.