Laser-Induced DNA Damage in U2OS Cells

U2OS cells stably expressing GFP-ATR were seeded into 24-well plates with a glass-bottom (Cellvis) 24 h before laser micro-irradiation in a density of 6 × 105 cells/mL. After seeding the cells into the 24 well plates, the specimen was first placed on an equilibrated bench for 20 min at room temperature (RT) to ensure equal cell distribution and then placed into an incubator. CuET was added to cells 5 h before micro-irradiation in final concentrations of 250 nM and 500 nM. Twenty minutes before laser micro-irradiation, cells were pre-sensitized towards UV-A wavelength by 20 µM 8-Methoxypsoralen (8-MOP) and placed inside Zeiss Axioimager Z.1 inverted microscope combined with the LSM 780 confocal module. Laser micro-irradiation was performed at 37 °C via X 40 water immersion objective (Zeiss C-Apo 403/1.2WDICIII), using a 355 nm 65 mW laser set on 100% power to induce the DNA damage. The total laser dose that can be further manipulated by the number of irradiation cycles was empirically set to two irradiation cycles. Subsequent immunofluorescence detection and quantitative analysis of the striation pattern in photo-manipulated samples were essentially performed as described previously [21 (link)].