Competitive Repopulation Assay for Murine Hematopoietic Stem Cells

CD45.2 mice were exposed to a sublethal dose (6.0 Gy) of TBI or sham irradiation and then treated with vehicle or ABT263 as shown in Figure 3a,g. BMCs were isolated and mixed with 2 × 10⁵ competitive BMCs pooled from three CD45.1 mice. The cell mixture was then transplanted into lethally irradiated (9.5 Gy TBI) CD45.1 recipients via retro-orbital injection of the venous sinus. For the normal aging study, 50 freshly sorted LT-HSCs from young and aged C57BL/6 mice treated with vehicle or ABT263 were mixed with 3 × 10⁵ competitive BMCs pooled from three CD45.1 mice, and the cell mixture was then transplanted into lethally irradiated CD45.1 recipients via retro-orbital injection of the venous sinus. Donor-cell engraftment in the recipients was analyzed at various times after transplantation as previously described^{4 (link),42} . For secondary BMT, 1 × 10⁶ BMCs were harvested from each primary recipient 4 months after primary BMT and were then transplanted into a lethally irradiated recipient as described above. Donor-cell engraftment in both primary and secondary recipient peripheral blood was analyzed as previously described^{4 (link),42} .