*2.5. Comet Assay*

Just after treatment (or after incubation for repair), HeLa cells were trypsinized and resupended in PBS (1 × 106 cells/mL). Thirty μL of each cell suspension were mixed with 140 μL of 1% low melting point agarose, and two drops of 70 μL were spread onto microscope slides precoated with 1% of normal melting point agarose. Glass cover slips were placed on the drops of agarose, which were allowed to set at 4 °C. Then the cover slips were removed and the cells embedded in agarose were lysed for 1 h by immersion in 2.5 M NaCl, 0.1 M Na2EDTA, 0.1 M Tris base, pH 10 and 1% Triton X-100 at 4 °C (lysis solution). For measurement of SBs, the slides were then placed in a horizontal gel electrophoresis tank and the DNA was allowed to unwind for 40 min in freshly prepared alkaline electrophoresis solution (0.3 M NaOH and 1 mM Na2EDTA, pH > 13).

For measurement of oxidized purines, after lysis, slides were washed three times (5 min each time) with buffer F (0.1 M KCl, 0.5 mM Na2EDTA, 40 mM HEPES, 0.2 mg/mL BSA, pH 8.0) and incubated for 30 min at 37 °C with FPG in buffer F, or with buffer F alone, in a moist box. After incubation the slides were placed in the electrophoresis solution.

Electrophoresis was carried out in the alkaline solution for 30 min at 1.1 V/cm and approximately 300 mA at 4 °C. The slides were washed in 0.4 M Tris base (pH 7.5) for 10 min at 4 °C to neutralize the excess alkali and 10 min in water at 4 °C. Then they were left to dry overnight.

Gels were stained with 25 μL of DAPI (4′,6-diamidine-2-phenylindol dihydrochloride, 1 μg/mL), covered with a cover slip and coded before microscopic analysis. DAPI-stained nuclei were evaluated with a Nikon Eclipse TS-100 fluorescence microscope. A total of 50 comets on each gel were visually scored as belonging to one of five classes according to the tail intensity. Each comet class was given a value between 0 and 4: (0) = undamaged and (4) = maximum damage [12]. The total score in arbitrary units (AU) was calculated by the following equation:

(percentage of cells in class 0 × 0) + (percentage of cells in class 1 × 1) + (percentage of cells in class 2 × 2) + (percentage of cells in class 3 × 3) + (percentage of cells in class 4 × 4).

Consequently, the total score was in the range from 0 to 400 arbitrary units. Net H2O2-induced damage was calculated by subtracting the comet score for non-H2O2-treated cells from the score + H2O2. Net FPG-sensitive sites were calculated by subtracting the score for the slide incubated with buffer from the score for the slide incubated with enzyme. Net Ro-induced damage was calculated as the difference in net FPG-sensitive sites between Ro-treated cells and cells not treated with Ro.
