4.3. Oil Red O Staining

SZ95 sebocytes were washed with phosphate-buffered saline (PBS) and fixed in 10% formaldehyde for 10 min. Fixed cells were stained for 30 min with filtered 0.7% Oil Red O solution (Sigma, St Louis, MO, USA) in propylene glycol. Stained cells were washed with distilled water, counterstained with hematoxylin, and visualized using microscopy. For quantitative analysis of Oil Red O staining, the area of lipid droplets was measured using the Image J software and normalized by the number of nuclei. For quantitative detection of intracellular lipids, Oil Red O was eluted by incubating cells with isopropanol for 10 min. Supernatant Oil Red O levels were determined by measuring the optical density at 500 nm using a microplate reader. To calculate the lipid amount per cell, the optical density value was normalized to the cell count and measured using an ADAM-MC automatic cell counter and the ADAM-MC software (Bulldog Bio, Portsmouth, NH, USA).