Human recombinant insulin

Human MDA-MB-231 BrCa cells were a gift from Dr. Danny Welch (University of Kansas Cancer Center). MDA-MB-231-GFP cells, analogous to the parental MDA-MB-231 cell line, were engineered to express green fluorescent protein (GFP). Both cell types were maintained in DMEM without sodium pyruvate plus 5% (v/v) FBS, 1% penicillin 100 U/ml/streptomycin 100 μg/ml and 1% non-essential amino acids. MCF-7 human ER+ BrCa cells (31 ), were a gift from Dr. Mark Kester (University of Virginia). MCF-7 cells were maintained in EMEM (Gibco) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin/100 mg/ml streptomycin, and 0.01μg/ml of recombinant human insulin (MP Biomedicals, Solon, OH). All BrCa cells were cultured in a humidified chamber with 5% CO₂ at 37 °C.
Murine MC3T3-E1 osteoblasts, a gift from Dr. Norman Karin, Roswell Park Cancer Institute, were maintained in alpha-MEM plus 10% FBS and 1% penicillin/streptomycin, and cultured in a humidified chamber of 5% CO₂ at 37 °C. MC3T3-E1 cells were seeded at 10,000 cells/cm² in 96-well plates. 24h later, growth medium was replaced with a differentiation medium consisting of αMEM plus 10% (v/v) FBS, 1% penicillin/streptomycin, 10mM β-glycerophosphate, and 50μg/mL ascorbic acid. MC3T3-E1 cells were differentiated for 21 days, and medium replaced every 3^rd day.