Serially transplantable and long-term BrdU-retaining HSCs do not uptake Ho in vivo. (A) Donor B6.SJL CD45.1⁺ mice were retro-orbitally injected with Ho 10 and 5 minutes before tissue sampling, and BM leukocytes were stained for lineage, CD41, Sca-1, KIT, CD48, and CD150 surface antigens. Gated LSK CD41⁻CD48⁻CD150⁺ phenotypic HSCs were sorted according to Ho uptake into Ho^neg and Ho^med described in Figure 1E. Each lethally irradiated congenic primary recipient (CD45.2⁺) was injected retro-orbitally with 50 sorted LSK CD41⁻CD48⁻CD150⁺ Ho^neg (Ho^neg) cells or 50 sorted LSK CD41⁻CD48⁻CD150⁺ Ho^med (Ho^med) cells together with 200 000 competitive CD45.2⁺ BM cells. Sixteen weeks after transplantation, CD45.1⁺ donor contribution was analyzed in blood CD45⁺ leukocytes, CD11b⁺ myeloid cells, B220⁺ B cells, and CD3⁺ T cells (top). Primary recipients were then killed, and BM leukocytes were harvested. CD45.2⁺ lethally irradiated secondary recipients were then transplanted with 2 × 10⁶ unmanipulated BM cells from the primary recipients. Sixteen weeks after transplantation, CD45.1⁺ donor contribution was analyzed in blood CD45⁺ leukocytes, CD11b⁺ myeloid cells, B220⁺ B cells, and CD3⁺ T cells (bottom). Each dot is the result from one mouse; bars are the average for each group. Significant differences are shown in each panel. (B) Mice were fed for 2 weeks with BrdU and then chased for 70 days. BrdU content was then measured by flow cytometry on sorted LSK CD41⁻CD48⁻CD150⁺ Ho^neg and LSK CD41⁻CD48⁻CD150⁺ Ho^med HSCs. The 2 histograms on the left and middle are overlays of BrdU stain on phenotypic LSK CD41⁻CD48⁻CD150⁺ HSCs from a control mouse without BrdU (shaded gray), Ho^neg (gray), and Ho^med (black) obtained from a Ho-perfused mouse. The 2 histograms are from 2 representative Ho-perfused mice. The right plot is the percentage of BrdU⁺ cells among Ho^neg (●) and Ho^med (○) LSK CD41⁻CD48⁻CD150⁺ HSCs. Each dot represents a separate mouse.