*4.4. Cell Culture and MTT Assay*

Human liver cancer cell lines (HepG2) and a normal fibroblast NIH-3T3 cell line (both cell lines procured from NCCS, Pune, India) were seeded in 96-well plates with a cell population of 1 × <sup>10</sup> <sup>4</sup> cells/well in DMEM/F12 with antibiotic solution and 10% FCS (Invitrogen, CA, USA). Cells were incubated in a 5% CO2 chamber at 37 ◦C. The monolayer cultured cells were washed with PBS, and then were treated with EO (10–100 μg/mL) with various test concentrations of tested samples in serum-free media and incubated for 24 h. The medium was aspirated, 0.5 mg/mL of MTT reagent was added, and the solution was incubated at 37 ◦C for 4 h. After the incubation period, measurements were carried out according to the method protocol described by Khalil et al. (2021) [5].
