2.7. Genotoxicity Assay

Micronuclei, which represent chromosomal fragments as a result of DNA breakage or entire chromosomes, were examined using the micronucleus analysis kit [32], according to the manufacturer’s instructions. Caco-2 cells were incubated for 24 h and LT97 cells for 48 h with ZnO NP or ZnCl₂ to allow most of the cells to divide, since micronuclei are formed during cell division. Vinblastine (25 ng/mL) and mitomycin C (2.5 µg/mL) were used as positive controls. After incubation, the plates were placed on ice for 20 min. A nucleic acid dye working solution was added, and the plates were placed on ice for 30 min under a light source to start the photolysis of ethidium monoazide. After removing the solution, complete lysis solution 1 (containing SYTOX^® Green nucleic acid stain and RNase) was added and incubated for 1 h in the dark at 37 °C. Finally, complete lysis solution 2 (containing SYTOX^® Green nucleic acid stain and green fluorescent beads) was added for 30 min at room temperature in the dark. The cells were measured using a flow cytometer (BD FACSCanto II; BD Biosciences, San Jose, CA, USA).