Cellstar 96 well plates

The HepG2 cell line (ATCC Cat# HB-8065, RRID:CVCL_0027) (CLS Cell Lines Service, Eppelheim, Germany) was cultured in DMEM high-glucose medium (Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated (30 min; 56 °C) FBS (formerly Sigma-Aldrich/now: Merck, Darmstadt, Germany), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM l-glutamine, seeded at 3.5 × 10⁴ cells/cm² in cell culture flask, or 6-well, or CellStar 96-well plates (Merck, Darmstadt, Germany) according to the experimental set-up and incubated for 24 h at 37 °C, 5% CO₂, 95% relative humidity. Subsequently, HepG2 cells were handled as detailed below under Section 2.5LOLA Treatment in Models of Steatosis, Insulin Resistance, and Metabolic Syndrome. (All other reagents from, formerly, Gibco/now: ThermoFisher Scientific, Oberhausen, Germany).

The cell viability in the Human breast cancer cell line (MCF-7) was determined by the MTT assay as previously described [24 ,25 (link)]. The cells were cultured and scattered in 96-well cell culture plates (Greiner CELLSTAR^® 96-well plates, Merck) at a 2 × 10⁶ cells/well concentration. Different concentrations of LTZ and LTZ-GA-AuNPs were added to the wells seeded with cells. The plates were incubated for 48 h. Consequently, the sample-laden medium was replaced with 100 μL of fresh culture medium and 20 μL of MTT solution (5 mg/mL in PBS) in each corresponding well. An aliquot (85 μL) from the wells was removed, and 50 μL of DMSO from each well was added and wholly mixed with the pipette and further incubated at 37 °C for 10 min. From the microplate reader (Spectrostar Nano, BMG Labtech, Ortenberg, Germany), cell viability was determined at 540 nm, which is based on the ability of viable cells to decrease the tetrazolium component of MTT into purple-colored formazan crystals [31 (link)]
The % cell viability was calculated by Equation (3).

The IC₅₀ value was determined by using a linear regression equation, i.e., y = mx + c.
Here, y = 50, m, and c values were derived from the viability graph. A value of p < 0.05 was considered statistically significant.