Laser microirradiation and imaging of cells

U2OS cells with transfection of green fluorescent protein (GFP)–tagged XRCC1, CHFR, and APLF were plated on glass-bottomed culture dishes (MatTek Corporation) and pretreated with or without 100 nM COH34 at 37°C for 1 hour before treatment with laser microirradiation. Laser microirradiation was performed using an IX 71 microscope (Olympus) coupled with the MicroPoint laser illumination and ablation system (Photonic Instruments Inc.). A 337.1-nm laser diode (3.4 mW) was transmitted through a specific dye cell and then yielded a 365-nm-wavelength laser beam that was focused through 603 UPlanSApo/1.35 oil objective to yield a spot size of 0.5 to 1 mm. The duration of cell exposure to the laser beam was ~3.5 ns. The pulse energy was 150 mJ at 10 Hz. Images were taken by the same microscope with the cellSens software (Olympus). GFP fluorescence at the laser line was converted into a numerical value using ImageJ. Normalized fluorescent curves from 50 cells from three independent experiments were averaged. The error bars represent SD.

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