In vivo Ho uptake by phenotypic BM endothelial cells, MSCs, and osteoblasts. 129SvJ mice were perfused with Ho dye intravenously 10 and 5 minutes before tissue sampling, hind limb bones were taken and crushed on ice in the presence of verapamil and reserpine, and BM cells were removed by several washes. Endosteal cells were then isolated by incubating crushed bones with collagenase in the presence of verapamil and reserpine. Cells were then stained with CD45, lineage, CD31, Sca-1, and CD51 antibodies. (A) Gating of CD45⁻Lin⁻ nonhematopoietic cells. (B) Gating of CD31^bright endothelial cells and CD31⁻ cells from the CD45⁻Lin⁻ gate in panel A. (C) Gating of Sca-1⁺CD51⁺ MSCs and Sca-1⁻CD51⁺ osteoblast-lineage cells from the CD45⁻Lin⁻CD31⁻ gate defined in panel B. (D-F) Representative dot plots of Ho blue fluorescence versus Ho red fluorescence of gated viable CD45⁻Lin⁻CD31^brightSca-1^bright BM endothelial cells, CD45⁻Lin⁻CD31⁻Sca-1^brightCD51⁺ MSCs, and CD45⁻Lin⁻CD31⁻Sca-1^brightCD51⁺ osteoblast lineage cells, respectively. (G) Distribution of Hoechst bright, medium, and negative cells among BM endothelial cells, MSCs, and osteoblasts and (H) mean fluorescence intensity of Ho blue fluorescence for BM endothelial cells and MSCs. Data are average ± SD from 3 mice.