Tcs sp5 dmi 6000b confocal microscope

MCF7 and MDA-MB-231 cells were grown on uncoated glass coverslips, placed in 60-mm plates. Cells were prepared as previously reported [49 (link)]. Fluorescence images were captured with a LEICA TCS SP5 DMI-6000B confocal microscope (Leica), using the following lasers: Argon (488 nm), DPSS (561 nm) and UV Diode (405 nm). Fluorescence images were analyzed with LEICA LAS AF (Leica) and ImageJ (NIH, http://rsb.info.nih.gov/ij) software.

TUNEL assay (TdT-mediated dUTP Nick-End Labeling) (Roche) was used to label free DNA-ends in damaged DNA in cells. The detection is based on the binding of fluorescein-12-dUTP to the 3′-OH of the DNA strand and was detected by a fluorescence microscope. Briefly, cells were cultured on glass coverslips and fixed with 3% PFA in PBS for 15 min at room temperature. PFA was removed and 200 mM glycine was added for 15 min. Cells were permeabilized with 0.2% triton X-100 for 15 min and blocked with 1% BSA in PBS for 30 min at room temperature or overnight at 4°C. Coverslips were incubated with 50 μl of TUNEL reaction mixture (prepared according to the manufacturer instructions) for 1 h at 37°C in darkness, followed by three washes for 10 min in PBS. Nuclei were stained with DAPI (Sigma), diluted 1:1,000 in PBS, for 10 min at room temperature, and washed three times for 10 min in PBS. Coverslips were mounted with Mowiol (Calbiochem-Merck, Darmstadt, Germany) on microscope slides. Samples were visualized using a Leica TCS SP5 DMI-6000B confocal microscope (Leica) and analyzed with the ImageJ software. As western blots and immunofluerescences, TUNEL assays were independently performed in triplicate and one representative experiment for each cell line was selected for the main figure. The number of cells in each independent experiment is indicated in the corresponding figure.