Rmscf

BM cells flushed from the femurs of the indicated mouse strain 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) progenitors were distinguished by examining the morphology of colonies. Total number of colonies per femur were calculated.

Freshly sorted cells were plated at 200 cells/ml in 1% methylcellulose culture medium supplemented with 30% FBS (Fisher Scientific), 2 mM L-Glutamine (Lonza), 0.1 mM hemin (Sigma-Aldrich), 50 ng/ml rmSCF (R&D Systems), 1 U/ml recombinant human erythropoietin (rhEPO, Amgen), and 5% vol/vol pokeweed mitogen mouse spleen cell conditioned medium. Cells were cultured at 5% CO₂, 5% O₂ in a humidified incubator, and colonies were scored on day 6 based on morphologies of CFU-GM, BFU-E, and CFU-GEMM progenitors. For high specific activity tritiated thymidine kill assay to determine % CFU in S-phase of the cell cycle, cells were incubated in IMDM media containing either control diluent or 50 mCi high specific activity tritiated thymidine for 30 minutes in the same incubator for cell culture aforementioned before washing and plating. For hCB CFU assay, cells were plated in 1% methylcellulose culture medium supplemented with 30% FBS, 2 mM L-Glutamine, human recombinant EPO (1U/ml), SCF (50 ng/ml), GM-CSF (10 ng/ml) and IL-3 (10 ng/ml) and cultured under the same conditions as for mouse cells. Human CFU-GM and CFU-GEMM colonies were scored on day 14 after culture, as this combination of growth factors does not pick up BFU-E.

Sorted pro-B or fraction B-cells from the bone marrow were cultured in pro-B medium (Opti-MEM [Invitrogen] supplemented with 10% fetal bovine serum [FBS], 50 μM β-mercaptoethanol, 2 mM L-Glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and indicated concentrations of recombinant murine (rm) IL-7 (Peprotech). Lineage-negative bone marrow cells were prepared with a lineage-depletion kit (Miltenyi) and cultured in Iscove’s Modified Dulbecco Medium (IMDM) with 10% FBS, 50 μM β-mercaptoethanol, 10 mM HEPES, 10 μM non-essential amino acids, 2 mM L-Glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Mediatech), as well as 50 ng/mL rm SCF (R&D System), 40 ng/mL rm Flt3L, and 5 ng/mL rm IL-7 (all from Peprotech).