2.8. Determination of Lipid Accumulation through Oil Red O Staining in HepG2 Cells

To understand the changes occurring in control and treated cells due to the accumulation of lipids in the cells and to evaluate the reduction in ethanol-induced hepatic injury, Oil red O staining was performed. After 48 h of incubation, the control and treated cells were incubated for 1 hour at room temperature (RT) with 1 ml of 10% neutral buffered formalin. The cells were then rinsed twice with milli-Q. Oil red O stain (stock solution: 0.5% in 100 ml isopropanol and 1 ml; working solution: 3 : 2 ratio diluted in milli-Q) was added to the cells. The cells were incubated at RT for 15 mins. The cells were washed with milli-Q twice to eliminate the excessive stains. The cells were then counterstained with Cole's hematoxylin solution for 30-45 min. The additional stain was removed by washing the cells with milli-Q. Cells were observed and snapped under an inverted microscope (Magnus, India) using PBS as a mounting medium.

Oil red O stain extraction by isopropanol method: to determine the difference in the lipid accumulation between control and treated cells, after staining the cells with Oil red O, the stain was extracted with absolute isopropanol. The extracted Oil red O stain was measured spectrophotometrically at 570 nm using isopropanol as blank [28].