*2.4. Treatment of Cells for Cellular Repair Assay*

Repair of DNA damage can be studied by treating cells with a DNA-damaging agent, incubating, and measuring the damage remaining at intervals. Using this cellular repair assay we investigated the kinetics of DNA SB rejoining and base excision repair (BER). To check the influence of vitamin C on the repair of SBs, HeLa cells were pre-incubated with 0, 50 or 100 μM vitamin C overnight. Then cells were washed with PBS, treated with 0 or 100 μM H2O2 for 5 min on ice, and incubated for 10, 30 or 60 min in culture medium. To check the influence of vitamin C on the repair of oxidized bases, pre-incubation overnight with 0, 50 or 100 μM vitamin C was followed by a wash with PBS, treatment with 0 or 1 μM Ro plus 5 min of light, and incubation for 1, 2, 4, 6, 8, or 24 h in culture medium. In another experimental design, HeLa cells in culture medium were pre-incubated with a higher concentration (200 μM) of vitamin C but for only 30 min. Then cells were washed with PBS, treated with 1 μM Ro plus 2.5 min of light, and incubated for 3 h in culture medium including 0 or 200 μM vitamin C. They were then washed with PBS and incubated again in culture medium with the same concentration of vitamin C for a further 3 h. The comet assay was performed after each time of incubation. The concentration of vitamin C used in these experiments was not genotoxic. Three independent experiments were performed.
