Adipogenic differentiation medium b

BMSCs in passage 2 were replated in the standard medium at 1×10⁵ cells/cm² in 6-well plates. Cells were incubated at 37°C in a 5% CO₂ humidified incubator. After the cells were 100% confluent, the standard medium was carefully aspirated off, and 2 ml Adipogenic Differentiation Medium A (induction medium) containing 10% FBS, 20 µg/ml insulin, 10 µM IBMX, 10 µM rosiglitazone, 10 µM dexamethasone, 1% penicillin/streptomycin, and 1% glutamine were added (Cyagen). Three days later, the Adipogenic Differentiation Medium A was replaced with Adipogenic Differentiation Medium B (maintenance medium), containing 10% FBS, 20 µg/ml insulin, 1% penicillin/streptomycin and 1% glutamine (Cyagen). Then, 24 h later, the medium was changed back to induction medium. To optimally differentiate BMSCs into adipogenic cells, the cycle of induction and maintenance was repeated three times. After three cycles, the cells were cultured in maintenance medium for an additional 7 days by replacing the medium every 3 days. After differentiation, Adipogenic Differentiation Medium was removed and the cells were rinsed twice with 0.1 M DPBS. Cells were fixed with 4% formaldehyde solution for 30 min. Then the cells were rinsed twice with 0.1 M DPBS and stained with oil red O (Cyagen) for 30 min at room temperature (RT). Cells were visualized under a light microscope and images were captured.

NP-MSCs from the second passage were seeded in a six-well plate at a density of 5 × 104 cells/cm². When the cells reached 100% confluence, the normal medium was replaced with adipogenic differentiation medium A (Cyagen, United States). After 3 days, differentiation medium A was replaced with adipogenic differentiation medium B (Cyagen, United States) for 24 h. Then, medium B was replaced with medium A again. This cycle was repeated for three to four cycles for a total of 28 days. After differentiation, the medium was removed from the wells, and the cells were washed with PBS and fixed with 4% paraformaldehyde solution for 30 min. After washing twice with PBS, the cells were stained with Oil Red O working solution (Cyagen, United States) for 30 min at room temperature. The culture was then washed three times with PBS and observed under an optical microscope.