Quantitative Lipid Droplet Analysis via Oil Red O Staining

Cytoplasmic lipid droplets were stained with oil red O, as described in a previous report [12 (link)]. Briefly, cells were rinsed three times in phosphate-buffered saline (PBS) and then fixed in 10% (v/v) formaldehyde for 10 min. The cells were washed twice with PBS, followed by staining for 30 min at 37℃ in freshly diluted oil red O (Sigma Chemical Co., St. Louis, MO, USA) solution (six parts oil red O stock and four parts H₂O; oil red O stock solution is 0.5% oil red O in isopropanol), followed by further washing with PBS. The stained cytoplasmic triglycerides were visualized and photographed using a microscope. For quantification of oil red O content, the cells were washed 3 times with distilled H₂O for removal of background staining, and isopropanol was added to resolve oil red O. The OD₅₁₀ nm of the de-staining isopropanol was measured by spectrophotometry.