Isolation and Culture of Murine Bone Marrow Stromal Cells

CD45⁻Ter119⁻CD31⁻ (TN) Scf-GFP⁺ stromal cells from digested BM were seeded in α-MEM media (Gibco) supplemented with 20 % fetal bovine serum (FBS; Gibco), 100 units/ml of penicillin and 100µg/ml of streptomycin, 10ng/ml of fibroblast growth factor (FGF)-basic (R&D Systems) and 2mM of L-glutamine (Thermo Fisher Scientific) or CFU-F culture media^{8 (link)} of phenol red–free α-MEM (Gibco) supplemented with 10 % FBS, 10 % MesenCult stimulatory supplement (STEMCELL Technologies), and 100 units/ml of penicillin and 100µg/ml of streptomycin. After 2–3 weeks of culture (passage 2–3), stromal cells were aliquoted and frozen with Recovery Cell Culture Freezing Medium (Gibco) and placed into liquid nitrogen. For control stroma, empty vector was infected into passage 2–3 stromal cells and cultured one passage prior to freezing into aliquots. For each experiment, we used these aliquots to minimize experimental variations.