Celltiter blue cell kit

Cell proliferation and viability assays were performed using a CellTiter‐Blue Cell Kit (Promega, Madison, WI). Cell cycle analysis was performed using flow cytometry cell cycle analysis with DAPI. The proliferation and differentiation potentials of MPCs were analyzed under 20% O₂ versus 1% O₂, in media containing 400 μM H₂O₂ (oxidative stress) for 3 hours, and in media containing 10 μM eeyarestatin for 6 hours to induce ER stress; and they were cultured in myogenic, osteogenic, and chondrogenic induction media. Chondrogenic (StemPro Chondrogenesis Differentiation Kit, Gibco) and osteogenic (DMEM, 10% FBS, 50 μg/ml ascorbic acid, 10 mM β‐glycerophosphate, 10 nM dexamethasone) induction media were used for the respective differentiation assays. Alkaline phosphatase (ALP) staining, computed tomography scanning of pellet cultures, and Alcian blue staining were used to confirm differentiation as described previously 56, 57.