Oil Red O Staining

Intracellular lipid accumulation was determined by Oil Red O staining. After removal of the culture media, the cells were washed twice with PBS, fixed with 4% PFA, and stained with Oil Red O (six parts 0.6% Oil Red O dye in isopropanol and four parts water) for 30 min. After rinsing three times with distilled water, the cells were photographed under a microscope.

To quantify lipid accumulation, lipids and Oil Red O were dissolved in isopropanol, and absorbance was measured by a microplate spectrophotometer at 500 nm. The percentage of Oil Red O-stained material relative to that in control wells was calculated as the absorbance at 500 nm (sample)/the absorbance at 500 nm (control) (Li et al., 2016).