To induce adipogenic differentiation, cells were stained with Oil Red O as previously described.22 (link) 1 × 104 cells cm−2 were cultured in 24-well plates in DMEM high glucose (Sigma, St. Louis, USA) supplemented with 10% FBS, 0.5 mmol l−1 isobutyl-methylxantine, 1 μmol l−1 dexame thasone, 5 μg ml−1 insulin and 150 μmol l−1 indomethacin (Sigma, St. Louis, USA). The cells were cultured, replacing the medium every 3 days. After 21 days of culture, the cells contained lipid droplets; they were fixed in a 10% solution of formaldehyde in aqueous phosphate buffer for about 1 h. Cells were stained with Oil red O solution.
To induce osteogenic differentiation, 1 × 104 cells cm−2 were cultured in 24-well plates in DMEM (Sigma, St. Louis, USA) supplemented with 10% FBS, 10 mmol l−1 β-glycerophosphate (Sigma, St. Louis, USA), 0.2 mmol l−1 ascorbic acid (Sigma, St. Louis, USA), and 100 nmol l−1 dexamethasone (Sigma, St. Louis, USA). Cells were cultured for 25 days, replacing the medium every 3 days. Then the cells were fixed with 10% formalin (Sigma, St. Louis, USA) for 15 min and assessed by Von Kossa staining.23 (link)
To induce osteogenic differentiation, 1 × 104 cells cm−2 were cultured in 24-well plates in DMEM (Sigma, St. Louis, USA) supplemented with 10% FBS, 10 mmol l−1 β-glycerophosphate (Sigma, St. Louis, USA), 0.2 mmol l−1 ascorbic acid (Sigma, St. Louis, USA), and 100 nmol l−1 dexamethasone (Sigma, St. Louis, USA). Cells were cultured for 25 days, replacing the medium every 3 days. Then the cells were fixed with 10% formalin (Sigma, St. Louis, USA) for 15 min and assessed by Von Kossa staining.23 (link)