3.3. Quantification of Lipid Droplets and Triglycerides in 3T3-L1 Cells

After treating 3T3-L1 cells for 12 days, the cells were carefully rinsed with PBS and fixed with 4% formaldehyde at room temperature for 1 h, followed by washing with 60% isopropanol. The 3T3-L1 cells were then stained with Oil Red O solution (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min and washed with 60% isopropanol and distilled water. Stained lipid droplets were quantified at an optical density of 492 nm using a microtiter plate reader (Allsheng, Hangzhou, China). For the quantification of cellular triglycerides, a triglyceride assay kit (Abcam, Cambridge, UK) was used. Briefly, after treating 3T3-L1 cells with BLs (0, 5, 10, or 20 μg/mL), the cells were washed with PBS and resuspended in 5% NP-40, followed by boiling for 5 min. The cell suspension was centrifuged at 13,000× g for 2 min and the supernatants were mixed with lipases to convert triglycerides into glycerol. After adding a triglyceride probe and a triglyceride enzyme mix, the mixture was incubated at room temperature for 1 h and the optical density was measured at 570 nm using a microtiter plate reader (Allsheng). The concentration of triglycerides was determined based on the standard curve of glycerol.