4.9. Measurement of DNA Damage by Alkaline Comet Assay

To detect cellular DNA damage as single-strand breaks, alkaline comet assay was performed using an Oxiselect Comet Assay® kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. In brief, L02 cells were pretreated with curcumin at 1.25, 2.5 and 5 μM for 2 h, then washed with PBS and exposed to FZD (40 μg/mL) or replaced with 0.2% DMSO (as the curcumin control) for 3 h. Cells in the negative control group were treated with 0.2% DMSO. To avoid artifacts resulting from necrotic and apoptotic cells, the cell suspensions (50 µL) were mixed with Hoechst 33342 (1 µg/mL) (Vigorous Biotechnology). After incubation in the dark for 30 min, necrosis and apoptosis were identified under a fluorescent microscope. In all groups, the cell viability was more than 90%. Then, the harvested cells were mixed with low melting point agarose and then transferred onto the CometSlide™ following solidification of cell-agarose mix. Cells were then incubated in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10 and 10% DMSO with 1% Triton X-100) in darkness at 4 °C for 1 h. After lysis, the slides were placed in alkaline solution (1mM Na₂-EDTA and 300 mM NaOH, pH 13) for 20 min at room temperature to allow DNA unwinding. Then, electrophoresis was performed in alkaline solution for 30 min at 25 V. After electrophoresis, the slides were washed twice for 5  min each at 4  °C in a neutralizing buffer (0.4 M Tris, pH 7.5), dehydrated in 70% ethanol, stained with Vista Green DNA dye (provided with the kit). Images were observed using fluorescent microscopy at an excitation wavelength of 490 nm and emission wavelength of 530 nm (Leica, Omachi, Japan). At least 100 randomly selected cells (30 or 40 cells from each of the three replicated slides) were analyzed using Comet Assay Software Project casp-1.2.2 (University of Wroclaw, Wroclaw, Poland). The tail length is the length of the tail (in pixels); the tail DNA% is calculated as (tail DNA intensity/cell DNA intensity) × 100; the tail moment length is the length from the center of the head to the center of the tail; and the olive tail moment is calculated as the tail moment length  ×  tail DNA%.