3h tdr

For HPC colony assays, BM cells flushed from femurs of indicated mice were plated at 5 × 10⁴ cells/mL in 1% methylcellulose culture medium with 0.1 mM hemin (Sigma-Aldrich; St. Louis, MO, USA), 30% FBS, 1 U/mL recombinant human erythropoietin (rhEPO; Amgen; Thousand Oaks, CA, USA), 50 ng/mL recombinant mouse stem cell factor (rmSCF; R&D Systems; cat. # 455-MC), and 5% vol/vol pokeweed mitogen mouse spleen cell conditioned medium. Colonies were scored after 6 days of incubation at 5% CO₂ and lowered 5% O₂ in a humidified chamber, and granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte burst-forming units (BFU-E), and granulocyte, erythrocyte, macrophage, megakaryocyte colony-forming units (CFU-GEMM) were distinguished by morphology of colonies. Total numbers of colonies per femur were calculated. For high specific activity tritiated thymidine kill assays, BM cells were treated with 50 μCi of high specific activity [3H]Tdr (20 Ci/mmol; DuPont NEN) at room temperature for 40 minutes then washed twice prior to plating in HPC colony assays. These assays were performed exactly as reported [14 (link)–16 ].

High specific activity tritiated thymidine ([3H]Tdr) kill assays were utilized to determine the percent HPC in cycle. BM cells were treated with 50 μCi of high specific activity [3H]Tdr (20 Ci/mmol; DuPont NEN) at room temperature for 40 minutes then washed twice immediately prior to use in HPC colony assays. The number of colonies in the [3H]Tdr treated plates was then compared to vehicle control treated plates and the percent colonies in S-phase was calculated.