2.5. Oil Red O Staining

To detect accumulation of intracellular neutral lipids, the control and HBE-treated HepG2 cells were fixed with 10% formalin for 1 h, and then stained with Oil Red O working solution for 30 min at room temperature. The cells were washed three times with deionized water and observed under an inverted microscope. For lipid quantification in HepG2 cells, 100% isopropanol was added to dissolve the Oil Red O reagent and the absorbance was measured at 500 nm. Further, HepG2 cells were incubated in 1mM FFA to induce hepatic steatosis for 24 h, with and without HBE supplementation. The cells were stained with Oil Red O solution and intracellular lipid accumulation was visually observed under a microscope (200×). Control cells were treated with 1% BSA only.