Oil Red O Staining for Adipocyte Lipid Content

The lipid content in the differentiated 3T3-L1 adipocytes was determined using the Oil Red O staining method, according to the method published by Kowalska et al. [21 (link)] with slight modifications. Briefly, the cells were washed with PBS after incubation, fixed in 250 µL formalin (10%) for 1 h, and washed with 60% isopropanol. The cells were then incubated with 100 µL Oil red O staining solution (0.21% in 60% isopropanol) for 10 min at room temperature, washed with water to remove unbound dye, and left to dry. Fat droplets, stained red, were extracted from cells using 375 µL 100% isopropanol, and the absorbance was measured at a wavelength of 520 nm with a microplate reader (Sirius HT, BioTek Instruments GmbH, Bad Friedrichshall, Germany). Resveratrol (c = 11.4 µg/mL and 22.8 µg/mL) was used as a positive control, according to Fischer-Posovsky et al. [26 (link)]. Lipid accumulation is presented as the percentage of the solvent control (DMSO; final concentration 0.1%).