2.5. Determination of Lipid Accumulation by Oil-Red O Staining

3T3-L1 cells, as described in Section 2.4, were incubated on a 12-well plate to induce adipocyte differentiation for 8 days. The Oil-Red O cell lipid index was obtained by rinsing the cells with normal saline on the eighth day, followed by fixing with 10% formaldehyde solution, and staining with Oil Red O solution (0.7 g in 200 mL isopropyl alcohol) at RT for 60 min. Following staining of lipid droplets, the staining solution was removed by rinsing with distilled water, followed by drying. Lipid droplets of 3T3-L1 adipocytes were observed under the microscope. Finally, the stain retained in the cells was eluted with isopropanol, and absorbance was measured at 520 nm using a microplate reader.