For HPC colony assays, BM cells flushed from femurs of the indicated mice were plated at 5 × 104 cells/mL in 1% methylcellulose culture medium with 0.1 mM hemin (MilliporeSigma), 30% FBS, 1 U/mL recombinant human erythropoietin (rhEPO) (Amgen), 50 ng/mL recombinant mouse stem cell factor (rmSCF) (R&D Systems; catalog 455-MC), and 5% vol/vol pokeweed mitogen mouse splenic cell conditioned medium. Colonies were scored after 6 days of incubation in 5% CO2 and lowered 5% O2 in a humidified chamber, and granulocyte-macrophage CFU (CFU-GM), erythrocyte burst-forming units (BFU-E), and granulocyte, erythrocyte, macrophage, and megakaryocyte colony-forming units (CFU-GEMM) were distinguished by morphology of colonies. The total numbers of colonies per femur were calculated. For high specific activity tritiated thymidine kill assays, BM cells were treated with 50 μCi high specific activity [3H]Tdr (20 Ci/mmol; DuPont NEN) at RT for 40 minutes and then washed twice prior to plating for HPC colony assays (62, 63). Note that this is currently the only means to assess the cycling status of functional populations of CFU-GM, BFU-E, and CFU-GEMM.

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