Cell differentiation was induced by addition of differentiation medium. Briefly, six wells of cells were transferred to differentiation medium I (growth medium plus 0.5 mmol/L 3-Isobutyl-1-methylxanthine (IBMX), 1 μmol/L dexamethasone (Dex, Dex medium has a weak ability to induce differentiation of 3T3-L1 cells into adipocytes), and 10 μg/mL Insulin (Promega, Heidelberg, Germany) 24 hours after transfection. Then, about 72 hours later, cells were rinsed twice with phosphate buffered saline (PBS), and differentiation medium I was replaced with differentiation medium II (growth medium plus 10 μg/mL Insulin). Differentiation medium II was renewed every 48 hours until the 12th day when the induction finished. The control group was treated as the same.
Lipid accumulation in adipocytes was visualized by staining with ORO (TaKaRa, Dalian, China). First, three wells of cells were rinsed twice with phosphate buffered saline (PBS) and fixed with 10% formaldehyde for 1 hour at room temperature. Then, after washing twice again with PBS, the cells were stained for at least 20 minutes in freshly diluted ORO solution (0.3% (w/v) ORO in 60% isopropanol). The staining solution was then removed, and cells were washed twice with PBS. ORO was extracted with isopropanol and the signal was quantified spectrophotometrically (NanoPhotometer, Implen Inc., CA, USA) at 530 nm54 (link).
Lipid accumulation in adipocytes was visualized by staining with ORO (TaKaRa, Dalian, China). First, three wells of cells were rinsed twice with phosphate buffered saline (PBS) and fixed with 10% formaldehyde for 1 hour at room temperature. Then, after washing twice again with PBS, the cells were stained for at least 20 minutes in freshly diluted ORO solution (0.3% (w/v) ORO in 60% isopropanol). The staining solution was then removed, and cells were washed twice with PBS. ORO was extracted with isopropanol and the signal was quantified spectrophotometrically (NanoPhotometer, Implen Inc., CA, USA) at 530 nm54 (link).