Elipse ts2r

Intracellular hydrogen peroxide and superoxide anion levels were evaluated using 2′,7′-dichlorofluorescin diacetate (DCFDA; Sigma-Aldrich, St. Louis, MO, USA) and dihydroethidium (DHE; Sigma-Aldrich, St. Louis, MO, USA). Briefly, HepG2 cells were cultured in 6-well plates and exposed to 20, 40, and 80 µM TEB or DMSO for 24 h with or without pretreatment with Lipofermata (20 µM in DMSO) for 1 h (n = 3 wells/group). After treatment with 10 µM DCFDA and DHE, the cells were incubated at 37 °C for 30 min in a 5% CO₂ incubator. After washing thrice with PBS, green fluorescence (DCFDA-positive) and red fluorescence (DHE-positive) areas were imaged using a Nikon Eclipse Ti2-U and Nikon Elipse Ts2R camera. Fluorescence intensities were quantified using ImageJ software version 1.52a (National Institutes of Health, Bethesda, MD, USA).

Intracellular neutral lipid such as triglycerides and cholesterol esters were evaluated using Oil red O dye (Sigma-Aldrich, St. Louis, MO, USA). HepG2 cells were seeded at a density of 2 × 10⁵ cells/well into 6-well plates and treated with 80, 160, and 320 µM FTF or 0.008% DMSO for 24 h (n = 3 wells per group). The cells were fixed with 10% formalin of 2.4 mL for 1 h at room temperature, and the fixed cells were rinsed with 60% isopropanol. Then, Oil red O staining solution of 1 mL was added to each well. After incubation for 10 min, the cells were washed with deionized distilled water thrice to remove the unbounded dye. Images were visualized and captured using a Nikon Eclipse Ti2-U and Nikon Elipse Ts2R camera (Nikon Co. Ltd., Tokyo, Japan). Then, the images were quantified using ImageJ software (National Institute of Health, Bethesda, MD, USA).