*4.6. Comet Assay*

The assay was carried out in alkaline conditions to detect SSB, DSB, and alkali-labile sites [80–82]. The slide's surface was pretreated with 1% normal melting point agarose (Sigma, St. Louis, MO, USA) in PBS. After a 45 min treatment, the cells were washed with PBS, incubated with trypsin-EDTA for 5 min at 37 ◦C, centrifuged, and resuspended in PBS; then, 20 µL of cell suspension was mixed with 80 µL of 0.75% low-melting agarose (Sigma, St. Louis, MO, USA) in PBS at 37 ◦C. Immediately, an 80 µL volume of the suspension was placed on a slide, covered with parafilm, and kept at 4 ◦C (10 min). The parafilm was removed and the slide was immersed in a cold lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, and 8 g of NaOH/890 mL of water, adjusted to pH 10, to which was added 1% Triton-X-100 and 10% DMSO an hour before use) and kept at 4 ◦C (from 1 to 15 days). The slides were incubated in cold electrophoresis buffer (300 mM NaOH and 1 mM EDTA at pH 13) for 20 min at 4 ◦C to unwind the DNA strands and expose the alkali-labile sites. Electrophoresis was performed at 25 V for 20 min. The buffer volume was adjusted to achieve a current intensity in the range of 250 to 300 mA. After that, the slides were washed with neutralization buffer (0.4 M Tris-HCl, pH 7.5) three times for 5 min each and washed with distilled water. Subsequently, the slides were stained with 80 µL of DAPI (6 µg/mL) (10 min), washed with distilled water, and covered with coverslips.

Comets were blind-counted under epifluorescence and representative photographs were taken with the confocal microscope in non-confocal conditions (confocal aperture 5) under a 20× objective and classified according to the degree of damage in five categories: from 1 to 5 (α = degree of damage). The following criteria were used to assess the degree of damage: 1: no damage and intact or with a halo surrounding the core; 2: a little damage (the DNA was distributed in an oval); 3: the anterior–posterior axis measured twice the diameter; 4: the compact DNA was reduced and a large cloud of DNA (long tail of the comet) appeared; and 5: the tail was separated from the rest of compact DNA.

A double-blind count of 100 cells was performed. A damage index (DDI) was calculated using the equation: *DDI* = Σ(*n*.α), where α (which can range from 1 to 5) expresses the degree of damage and *n* is the number of cells with the degree of damage α.
