*2.2. Cell Treatment for DNA Damage and Protection*

HeLa cells in culture medium were incubated with 0, 5, 25, 50, 100 or 200 μM vitamin C (ascorbic acid) for 30 min or 6 h (3 h incubation, followed by a wash with PBS and a further 3 h incubation with vitamin C at the same concentration) at 37 °C in the dark. The two consecutive 3 h treatments were designed to allow for the possible instability of vitamin C in solution. Vitamin C was dissolved in PBS in the dark just before use in each experiment. After treatment the comet assay was performed as described below. To check for DNA protection, after vitamin C treatment, cells were washed with PBS and then treated on ice with 25 μM H2O2 for 5 min to induce SBs, or with 1 μM of the photosensitizer Ro (Ro-19-8022, from F. Hoffmann-La Roche) plus 1.5 min visible light (500 W tungsten halogen lamp, at 33 cm on ice) to induce oxidized purines, mostly 8-oxoGua. After treatment the comet assay was performed (see below). Three independent experiments were carried out.
