Cells were seeded on culture slides. Cells were pulse labeled with 10 µM EdU for 20 minutes prior to treatment. After treatment the cells were fixed, permeabilized and blocked. Foci were detected using anti-53BP1 (Rabbit-anti 53BP1, 1:2000, Novus Biologicals), RPA (Mouse-anti RPA, 1:400, Santa Cruz), yH2AX (Rabbit-anti yH2AX, 1:250, Novus Biologicals), RAD51 (Rabbit, 1:500, Calbiochem), IFN-ß1 (Rabbit-anti IFN-ß1, 1:1000, Cell signaling) or IRF3 (Rabbit-anti IRF3, 1:400, Cell Signaling) followed by Alexa Fluor 488 goat anti rabbit IgG (Cell Signaling, 1:600), AlexaFluor 488 goat anti mouse IgG (Cell signaling, 1:500), AlexaFluor 594 goat anti rabbit IgG (Abcam, 1:600) or AlexaFluor 647 goat anti rabbit IgG (Cell Signaling, 1:600) and mounted (Vector Laboratories). EdU was stained with Alexa Fluor Azide 594 (Life Technologies, 1:500) and nuclei were stained with DAPI. Foci and fluorescence Intensity were quantified manually by capturing fluorescence images using a Zeiss Axioplan 2 fluorescence microscope equipped with a charge-coupled device camera and Axiovision software followed by quantification by Image J software. RPA/yH2AX-Foci were quantified automatically by the Aklides®-system (Medipan). Foci and fluorescence intensities of 100 cells per dose per slide and experiment were quantified.
Meyer F., Engel A.M., Krause A.K., Wagner T., Poole L., Dubrovska A., Peitzsch C., Rothkamm K., Petersen C, & Borgmann K. (2022). Efficient DNA Repair Mitigates Replication Stress Resulting in Less Immunogenic Cytosolic DNA in Radioresistant Breast Cancer Stem Cells. Frontiers in Immunology, 13, 765284.
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