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Blood, Vol. 109, Issue 10, 4494-4502, May 15, 2007
BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors
Blood Perry et al.
109: 4494
Supplemental materials for: Perry et al, Vol 109, Issue 10, 4494-4502
Detailed methods of the culture conditions for grown spleen stress erythroid progenitors
Single-cell suspensions of splenocytes were prepared and plated in base media containing 15% fetal bovine serum, 1% bovine serum albumin, 10 µg/mL insulin, 200 µg/mL human transferrin (iron saturated), 10−4 M 2-mercaptoethanol, 2 mM L-glutamine, and 3 U/mL erythropoietin (R&D Systems, Minneapolis, MN) in IMDM. The cells were cultured at 20% or 2% O2, and were supplemented with 15 ng/mL BMP4 (R&D Systems) and/or 50 ng/mL SCF (PeproTech, Rocky Hill, NJ) as indicated for 5 days. The live cells were counted by Trypan Blue staining. Progenitor cell populations were analyzed by flow cytometry using APC-conjugated anti-Kit, FITC-conjugated anti-CD71, and PE-conjugated anti-Ter119 from BD Pharmingen (San Diego, CA) and Draq5 (Alexis Biochemicals, San Diego, CA). The percentage of dead cells was measured by propidium iodide staining. Single-cell suspensions were antibody labeled and washed with 2% fetal calf serum in PBS. Surface-labeled cells were analyzed using an FC500 flow cytometer (Beckman Coulter, Miami Lakes, FL) and CXP software.
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