Flow cytometric analysis of BrdU incorporation in donor Sca-1⁺ and Sca-1⁺lin⁻ cells in irradiated and nonirradiated recipient BM, and cell cycle analysis of donor Sca-1⁺ cells in irradiated recipient BM 6 hours after transplantation.

55 × 10⁶ LDBM cells of B6.BoyJ origin were transplanted into irradiated or nonirradiated C57Bl/6 mice. Mice were killed 6 hours later, and BM cells lysed and stained with CD45.1-PE, Sca-1–biotin, CD3-cychrome, B220-cychrome, followed by streptavidin-APC. Cells were permeabilized and stained with anti-BrdU–FITC as described in “Materials and methods.” (A) and (B) represent typical analyses where donor cells (5.5%) are gated from all nucleated cells and analyzed for Sca-1 and lineage expression (B). Donor Sca-1+ cells (27% of donor cells; solid region in B) were then analyzed for their BrdU incorporation in irradiated (D) or nonirradiated (F) mice, or cell cycle status by PI staining (typical cell cycle histogram shown in G). Donor Sca-1⁺lin⁻ cells (21% of donor Sca-1⁺ cells; dotted region in B) were likewise analyzed for BrdU incorporation (C,E). Sca-1⁺lin⁻histograms contain between 75 and 85 events, whereas Sca-1⁺histograms contain between 256 and 271 events. Frequencies of BrdU⁺ cells and cells in G0/G1 are given on each histogram.