Fresh oil red o solution

ASCs were characterized by measuring their colony forming, adipogenic and osteogenic abilities. ASCs at passage 3 were plated at a density of 100 cells per 10-cm dish and cultured in complete medium for 7 days. For the colony forming assay, the cells were stained with 0.5% crystal violet (Kanto Chemical, Tokyo, Japan) in methanol for 5 min and washed twice with distilled water. For the adipogenesis assay, the medium was changed to complete medium supplemented with 0.5 mmol/L dexamethasone (Fuji Pharma, Tokyo, Japan), 0.5 mmol/L isobutyl-1-methyl xanthine (Sigma–Aldrich, St. Louis, MO, USA) and 50 mmol/L indomethacin (Wako Pure Chemical Industries). After 21 days, the cells were fixed with 4% paraformaldehyde (Muto Pure Chemical, Tokyo, Japan) for 1 h and stained with fresh oil red O solution (Fujifilm Wako Pure Chemical Corporation) for 3 h. For the osteogenesis assay, the medium was switched to complete medium supplemented with 100 nmol/L dexamethasone (Fuji Pharma, Tokyo, Japan), 10 mmol/L β-glycerophosphate (Sigma–Aldrich) and 50 mmol/L ascorbic acid (Wako Pure Chemical Industries). After 21 days, the cells were stained with 1% alizarin red S solution (Wako Pure Chemical Industries).

ADSCs were characterized based on their adipogenic, osteogenic, and chondrogenic capacities. For the adipogenesis assay, the medium was changed to MSCgo adipogenic (Cosmobio, Tokyo, Japan). After 21 days, the cells were fixed with 4% paraformaldehyde (Muto Pure Chemical, Tokyo, Japan) for 1 h and stained with fresh oil red O solution (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for 3 h. For the osteogenesis assay, the medium was switched to MSCgo Osteogenic (Cosmobio, Tokyo, Japan). After 21 days, the cells were stained with 1% alizarin red S solution (Wako Pure Chemical Industries, Osaka, Japan). For the chondrogenic assay, the medium was switched to MSCgo chondrogenic (Cosmobio, Tokyo, Japan). After 21 days, the cells were stained with 0.2 mL of 1% Alcian Blue solution (Wako Pure Chemical Industries, Osaka, Japan).