Differentiation of 3T3-L1 Cells and Oil Red O Staining

The 3T3-L1 preadipocytes were cultured in high glucose DMEM supplemented with 10% bovine calf serum. Cells were split 1:10 every 2 days to prevent reaching confluency. Cells were incubated in 95% O₂ and 5% CO₂ at 37 °C. For adipocyte differentiation, the preadipocytes were allowed to grow to confluency in 10% bovine calf serum. Two days post confluency, cells were switched to DMEM supplemented with 10% fetal bovine serum (FBS) and stimulated with a differentiation cocktail (IDM) containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, and 1.5 μg/mL insulin. Two days after IDM induction, cells were fed with DMEM plus 10% FBS and 1.5 μg/mL insulin. Three days later, cells were cultured in DMEM plus 10% FBS and media was replaced every 2–3 days until full differentiation was achieved. For oil red O staining, a stock oil red O solution was prepared by adding 150 mg of oil Red O powder to 50 mL of isopropanol. Then, 3 parts of oil Red O stock solution were mixed with 2 parts distilled water and incubated for 10 min at room temperature. The oil red O working solution was filtered through Whatman filter paper. Differentiated 3T3-L1 adipocytes cultured on 6-well dishes were first fixed in 10% formalin for 30–60 min. Cells were then briefly rinsed with 3 times with distilled water and incubated in 60% isopropanol for 5 min. Freshly prepared oil red O working solution was added to the cells for 5 min. Cultures were rinsed with room temperature tap water until the water rinses were clear. The stained lipid droplets were photographed with a digital camera. Bright-field images were captured with the IX50 inverted system microscope (Olympus corporation of the Americas) using magnification of ×40. A non-stained area was chosen to set the white balance.