Indirect immunofluorescence (IF) and image analysis

For immunofluorescence analysis, cells were grown on poly-L-lysine (Biochrom) coated coverslips. For analysis using BrdU detection, cells were pre-incubated for 24 h in media containing 10 μM BrdU. When required, S-phase cells were labeled with 10 μM of 5-ethynyl-2′-deoxyuridin (EdU) for 30 min. After irradiation, cells were washed three times with ice-cold PBS and fixed in PFA solution, (3% paraformaldehyde and 2% sucrose), for 15 min at RT. Subsequently, cells were washed three times with PBS and permeabilized in P-solution (100 mM Tris pH 7.4, 50 mM EDTA, 0.5% Triton X-100) for 10 min at RT. After washing three times with PBS, samples were blocked in PBG solution (0.2% skin fish gelatin, 0.5% BSA fraction V, in PBS) overnight at 4 °C. Primary antibodies, pATM-S1981 (Cell Signaling Technology, 4526), pChk2-T68 (BIOZOL, BZL08950), Cyclin B1 (Santa Cruz Biotechnology, H-433), 53BP1 (Santa Cruz Biotechnology, H-300) and RPA70 (αSSB70B, mouse hybridoma cell line kindly provided by Dr. G. Hurwitz)¹¹⁸ were diluted (1:300) in PBG solution, while the anti-BrdU antibody (BD) was used at a dilution of 1:100. The cover slips were incubated at RT for 2 h and washed three times with PBS-T (0.05% Tween-20 in PBS). Alexa Fluor-conjugated secondary antibodies, anti-mouse IgG Alexa Fluor 488 and anti-rabbit IgG Alexa Fluor 568 (Thermo Fisher Scientific, A11001; A11011), were applied at 1:400 dilution for 1 h at RT. When necessary, the EdU signal was developed using an EdU staining kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Finally, cells were counterstained with 100 ng/ml DAPI (Thermo Fisher Scientific) at RT for 5 min and coverslips were mounted in PromoFluor antifade reagent (PromoCell). Scanning was carried out on a Leica TCS-SP5 confocal microscope (Leica Microsystems). For each slide, 5 fields were scanned (about 200 nuclei) and the Z-stacks were processed using Imaris image analysis software (Bitplane). Immunofluorescence analysis of EdU labeled cells was carried out using a high-content imaging and image analysis platform (Metasystems). In total ~1600 cells were analyzed to obtain 100–150 EdU-negative, G₂-phase cells for the quantification of parameters of interest.