8-Oxo-dG assay

A previously described assay based on the property of avidin to bind with high specificity to 8-oxo-dG was used for the 8-oxoG measurements [24]. Briefly, cells were fixed in methanol at −20 °C for 20 min and incubated for 15 min in TBS, 0.1 % Triton X-100. Blocking was performed in 15 % FBS, 0.1 % Triton X-100 in TBS for 2 h at room temperature (RT). Cells were then incubated with 10 μg/ml Alexa488-conjugated avidin (Invitrogen) in blocking solution for 1 h at 37 °C. Next they were rinsed twice in TBS, 0.1 % Triton X-100 for 5 min each round at room temperature. After a quick rinse in distilled water, DNA was counterstained with ToPro3-Iodide (LifeTechnologies) for 15 min at room temperature, followed by a final rinse in TBS.

Coverslips were mounted with ProLongGold (Invitrogen) and cells were observed under a Zeiss Axiolab fluorescence microscope equipped with a monochrome CCD camera. Analysis was conducted with NIH-imageJ, with respect to mean intensity in the nucleus (To-Pro3 served as a DNA reference). All experiments were repeated five times.