2.4. Screening by Oil-Red-O Staining

Oil-red-O staining was conducted to evaluate total lipid accumulation in PA-induced HepG2 cells [26,27]. HepG2 cells were seeded into 12-well plates at a density of 1 × 10⁵ cells/well and acclimatized for 24 h. Next, each LAB strain (10⁷ CFU/mL in fresh free antibiotic MEM containing 0.75 mM PA) was added. Wells treated with PA solution alone were used as negative controls. After 24 h, cells were washed twice with PBS and fixed at 4 °C for 5 min with 10% formalin solution (Cat. HT501128, Sigma Aldrich, St. Louis, MO, USA). The formalin solution was then removed and cells were fixed for 1 h in 10% formalin solution. The fixed cells were then washed twice with distilled water and once with 60% isopropanol, and then dried for 5 min. After treatment with Oil-red-O solution, the dried cells were incubated for 10 min in a dark room. The Oil-red-O stock was prepared by dissolving 0.5 g of Oil-red-O (Cat. O0625, Sigma Aldrich, St. Louis, MO, USA) in 100 mL of isopropanol. The Oil-red-O stock was then mixed with isopropanol at a ratio of 6:4 for use in the experiments. The stained cells were washed twice with distilled water and dissolved in 100% isopropanol, and absorbance was measured at 520 nm.