Crystal violet

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).