The induction of adipogenic differentiation was initiated as previously described (41 (link)–43 (link)). Briefly, BM-MSCs (3×105 cells) were plated in 6-well plates and cultured in an adipogenic medium comprising DMEM supplemented with 0.5 mM 3-isobutyl-1-methyl-xanthine (Sigma-Aldrich; Merck KGaA), 10% FBS, 100 mg/ml indomethacin (MilliporeSigma), 0.01 mg/ml insulin (MilliporeSigma) and 1 mM dexamethasone (MilliporeSigma) at 37°C for 12 days. Adipogenic differentiation was analyzed via the detection of fatty acid-binding protein 4 (FABP4), lipoprotein lipase (LPL), perilipin-1 (PLIN1), peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) mRNA expression levels using RT-qPCR.
The accumulation of lipid droplets was measured using Oil Red O staining. Briefly, BM-MSCs were washed twice with PBS at the end of the adipogenic differentiation. Subsequently, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with Oil Red O staining solution (Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at room temperature. Images were captured using a light microscope.
The accumulation of lipid droplets was measured using Oil Red O staining. Briefly, BM-MSCs were washed twice with PBS at the end of the adipogenic differentiation. Subsequently, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with Oil Red O staining solution (Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at room temperature. Images were captured using a light microscope.
