MC3T3-E1 cell culture and osteoblast differentiation

Mouse MC3T3-E1 preosteoblasts (American Type Culture Collection) were cultured in α-MEM and 10% FBS. Osteoblast differentiation from MC3T3-E1 cells was induced by culturing them in α-MEM supplemented with 10% FBS, 10 mM β-glycerol phosphate, and 50 μM of ascorbate containing 200 ng/ml of IL-20 (R&D system), 2 μg/ml of 7E, or IL-20+7E for 14 days. The osteoblast differentiation medium was replaced once every 2 days. The osteogenic activity was evaluated using ALP staining 14 days. ALP activity was measured using an ALP assay kit 14 days after the cells had been cultured.