Oil red o solution

ATMSC was seeded at a number of 1×10⁵cells/well in a 4-well plate in MSC culture medium. After getting 100% confluency, the culture medium was changed to adipogenic or osteogenic differentiation medium. The culture medium was changed twice per week for 3 weeks. Adipogenic differentiation medium consisted of IMDM supplemented with 10% FBS, 0.1 mM dexamethasone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 2 mg/mL insulin (Wako), and 0.1 mM indomethacine (Sigma-Aldrich). The formation of adipocytes was examined by staining with Oil Red O solution (Muto Pure Chemicals, Tokyo, Japan). For quantification, cells were dissolved with 4% IGPAL CA630 (Sigma-Aldrich) in isopropanol, and the absorbance at 492 nm was measured. Osteogenic differentiation medium consisted of IMDM supplemented with 1% FBS, 0.1 mM dexamethasone (Sigma-Aldrich), 10 mM β-glycerol-2-phosphate (Sigma-Aldrich), 0.2 mM ascorbic acid (Sigma-Aldrich), and 50 ng/mL human EGF (Wako). The formation of mineralized matrix was examined by staining with Alizarin Red S (Kodak, Rochester, NY). For quantification, cells were dissolved with 0.2 N HCl (Wako) and 5% sodium dodecyl sulfate, then the absorbance at 480 nm was measured.

Triglyceride content in the livers was measured using a triglyceride colorimetric assay kit (Cayman Chem Inc., Ann Arbor, MI, USA). Oil Red O staining was performed on cryosections (8 μm) incubated in 60% isopropanol for 30 s and in the Oil Red O solution (Muto Pure Chemicals, Co., Ltd., Tokyo, Japan) at 37 °C for 10 min. Specimen integrity was verified by staining with Mayer’s hematoxylin for 2 min.