γH2AX biomarker-based genotoxicity assay

Genotoxicity of lilial and its metabolites was evaluated using HCS DNA Damage Kit (Invitrogen, Waltham, Massachusetts, USA). Briefly, HeLa and CHO-K1 cells were seeded at a concentration of 1 × 10⁵ cells/ml into the 96-well plates. CHO-K1 cells were used for comparison with HPRT test. HeLa cells were recommended by the manufacturer of the kit. After 24 h, cells were rinsed with PBS and cultivation medium (“Cell lines” Section) without ATB, with a reduced content of FBS (5%) was added. 30 µM valinomycin (in DMSO) was used as a positive control Samples were applied in the presence of non-toxic sample concentrations, i.e. 100 µM either with or without S9 mix. After 4 h or 24 h incubation, the cells were fixed and stained according to HCS DNA Damage Kit instruction using Hoechst 33342 (350/461 nm binds to DNA; cell count), Alexa Fluor^® 555 (555/565 nm secondary antibody; binds to primary antibody), an antibody against phosphorylated H2AX (Ser139, DNA damage, which is induced in response to double-strand break formation). Cells were photographed using a fluorescence microscope (Olympus IX83); filters: DAPI (500 ms) and Cy3 (500 ms). Bioimage analysis in ImageJ software was performed as described previously³⁴.