Evaluation of Lipid Droplet Modulation

HepG2 cells were seeded in 96-well plates at 4 × 10³ cells/well and adhered overnight. The medium was changed to DMEM supplemented with 62 µM sodium oleate (Sigma-Aldrich, St. Louis, MO, USA) to induce the presence of lipid droplets [42 (link)] and two different concentrations of plant extracts (final concentration of 30, 10 µg/mL; dilution 1:200 of stock concentration). After 6 h, cells were stained with 75 ng/mL Nile red (Sigma-Aldrich) and 10 µg/mL Hoechst 33342 (Sigma Aldrich) in HBSS. After incubating at 37 °C for 10 min and in the absence of light, cells were washed twice with HBSS. Fluorescence was read in a Synergy HT multi-detection microplate reader (Biotek) at 485/572 nm excitation/emission for Nile red and 360/460 nm for Hoechst. Following this, cells were fixed with ice-cold trichloroacetic acid (TCA) for 1 h at 4 °C in the dark and then washed four times with ddH₂O to remove all the TCA. After being air-dried, the cells were stained with sulforhodamine B (SRB) in 1% acetic acid for 15 min and rinsed quickly with 1% acetic acid five times to remove unbound SRB. Plates were air-dried and bound dyes were solubilized with 150 µL Tris–HCl (10 mmol/L, pH 10.5) before reading the absorbance at 492 nm with the reference at 650 nm in a multi-detection microplate reader (Biotek, Synergy HT).