2.3. Oil Red O Staining and Quantitation

On day 8, the cells were washed three times with phosphate-buffered saline (PBS) and then fixed for 30 min with 4% buffered paraformaldehyde. Then, cells were stained with Oil red O for 20 min following the manufacture's instruction. After washed three times with PBS, the lipid droplets were observed and photographed under a microscope (Axio Observer A1; Carl Zeiss, Göttingen, Germany). To measure the total content of lipids in each well, Oil-Red-O-stained lipids were dissolved in 100% isopropanol, and the absorbance of each sample was measured at 490 nm using a Multiskan GO microplate reader (Thermo Fisher, MA, USA).