4.4. LDL Uptake Assay

The assay was conducted as described previously [40] with slight modification. Briefly, HepG2 cells were maintained in MEM supplemented with 10% FBS. The cells were seeded in 96 well black plates at a density of 1 × 10⁴ cells per well and grown to 70% confluence. Then, cells were incubated with serum-free opti-MEM for 1 h, followed by incubation with 1 μM simvastatin and/or 5 μM lunasin for 20 h. Thereafter, 20 μg/mL Dil-LDL were added, and the cells were incubated in the dark for an additional 4 h. Cells incubated with opti-MEM without Dil-LDL were used as the negative control. Cells incubated with Opti-MEM and 20 μg/mL Dil-LDL were used as the control for normalization, respectively. After rinsing with PBS 3 times, LDL uptake was measured on a fluorescence plate reader (Varioskan flash, Thermo, Waltham, MA, USA) at an excitation wavelength of 520 nm and an emission wavelength of 580 nm.