Achroplan 63 0.9w dic 3 objective

The cells were prepared as described in spot assay. For visualizing DNA in living cells, 10 mg/ml DAPI was added to the culture 30 min before imaging. Next, 1 ml aliquots were washed, pelleted and resuspended in 200μl of fresh synthetic minimal medium (SD). Then 5 ml volume of cells were immobilized on a glass slide precoated with 0.1% poly-L-lysine solutions (Sigma), to prevent evaporation, the cover glass was sealed with Cytoseal™ XYL mounting medium (Richard-Allan Scientific). Cells were viewed under a Zeiss Axioplan 2 imaging microscope (Carl Zeiss, Thornwood, NY) with a water-immersion Achroplan 63_/0.9W/DIC III objective. The illumination source was a 100-W mercury arc lamp. Cell images were taken using an AxioCamHRm digital camera operated via AxioVision 4.5 software. Confocal images were captured with a LSM 510 META system operated via META 3.2 software. The wavelengths of the filters used to visualize the RAD52-GFP (excitation 488 nm; emission 509 nm) and 4',6-diamidino-2-phenylindole (DAPI; excitation 358 nm, emission 463 nm). Image acquisition times for RAD52-GFP was 1100 ms. At least 300 nuclei were counted for total of 6 – 10 field of cells, the observations of RAD52-GFP foci formation are from 1.5 to 6 hr after MNNG treatment, the results shown are based on 3 independent experiments. Unless otherwise noted, all experiments were performed at room temperature.