2.5. Staining of Lipid Droplets with Oil Red O and BODIPY 493/503

HepG2 cells (5 × 10⁵ cells/ml) were pretreated with various concentrations of ZA-M for 1 h before exposure of 500 μM OA for 24 h. After incubation, cells were fixed with 4% paraformaldehyde and stained with working solution of Oil Red O for 10 min at room temperature. After several washings, cells were observed under a light microscope. To quantify Oil Red O content, 100% isopropanol was added to each sample, which was read using a microplate reader at 500 nm. The cells were incubated for 15 min with Hoechst 33342 to stain nuclei and Bodipy 493/503 to stain neutral lipids and observed under a fluorescent microscope (Nikon, Japan).