Alizarin red staining

For assays of adipogenesis or osteogenesis, expanded single cells during passages three to five were seeded at the density of 2.5 × 104 cells/cm² in 24-well plates in DMEM with 10% FBS. At 90% confluence, the medium was switched to the adipogenesis differentiation medium or the osteogenesis differentiation medium, respectively (Invitrogen) and changed every 3 days. After 21 days of culturing, cells were fixed with 4% formaldehyde and stained with oil red O for adipocytes by adipogenesis Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) or with 2% Alizarin Red for osteocytes following the manufacturer's protocol. Cells with oil droplet stained by Oil Red were quantified by measuring at OD at 492 nm in triplicate cultures. Mineralized cells with positive Alizarin Red staining (Alfa Aesar, Tewksbury, MA, USA) were quantified by measuring OD at 405 nm in triplicate cultures.
For the chondrogenesis assay, pellets were prepared by spinning down 1 × 105 cells and incubating in a 15-mL conical tube in chondrogenesis differentiation medium (Invitrogen) with the medium changed every 3 days. After 28 days of culturing, cells were fixed with 4% formaldehyde, and stained with Alcian Blue (Merck, Darmstadt, Germany).