4.4. CHO-K1 Cytotoxicity Assay: Cell Viability Test

The Chinese hamster ovary CHO-K1 cell line was obtained from the American Type Culture Collection (ATCC CCL-61). The CHO-K1 cell line was cultured in a humidified incubator at 37 °C with 5% CO₂, in F-12K Medium (Kaighn’s Modification of Ham’s F-12 Medium) (ATCC) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and antibiotics (1% streptomycin/penicillin salts mixture, Sigma–Aldrich, Darmstadt, Germany).

In the assay the maximum non-cytotoxic amounts of test compounds JH3 and JH10 and commercial UV filter 4MBC were determined. The cells were seeded at a density of 6 × 10³ on 96-well plates. Following overnight culture, the cells were then treated with increasing doses of test compounds (1–150 µM) for 24 h. Test compounds and 4MBC were dissolved in DMSO (solvent, final concentration 0.1%) and ethanol (co-solvent, final concentration 1%). At the end of the incubation period, 10 µL of MTT reagent was added to each well. After 4 h incubation (37 °C, 5% CO₂), the medium was discarded, and the formazan produced in the cells appeared as dark crystals in the bottom of the wells. Next, pure DMSO was added to each well. The optical density data of converted dye was measured at 570 nm (A570) on microplate reader. Viability (percent of control) was determined by dividing A570 of experimental wells by A570 of control wells × 100% [15,77]. The experiment was conducted two times with three repetitions for each condition.