Quantitative Oil Red O Staining of Lipid Droplets

LDs in the cells were stained and quantified using Oil Red O staining, as previously described [22 (link)]. The Oil Red O solution constituted 0.3% Oil Red O/2-propanol:H₂O in the ratio 6:4 and was filtered through a 0.45 μm PVDF filter (Millipore, Burlington, MA, USA). Then, 5% v/v 60% isopropanol/H₂O was added to the filtered solution. Cells were washed once with PBS, followed by washing with 60% 2-propanol and then added to an Oil Red O solution for 30 min. After staining, the cells were washed thrice with 60% 2-propanol/H₂O. After drying the dishes overnight, extraction of Oil Red O was performed with 100% 2-propanol, once for 60 min and twice for 10 min. For quantification, the absorbance of Oil Red O extract was measured at 490 nm using a microplate reader.