Immunofluorescence staining and automatic nuclear foci quantification

In 12-well plates, approximately 100,000 EUFA1341 cells complemented with FLAG-PALB2 variants were seeded onto glass coverslips pre-coated, 5 min at room temperature, with 1 µg/ml PEI (408727, Sigma-Aldrich) in plain DMEM. The next day, the growth medium was refreshed with medium supplemented with 10 nM CPT in DMSO or the equivalent volume of vehicle. After 17h of incubation (37°C, 5% CO ₂), the cells were washed twice with 1x PBS and fixed with 4% formaldehyde (Pierce, 28908) in 1x PBS (15 min, RT). The cells were immediately incubated with 125 mM Glycine (Sigma-Aldrich, G7126) in 1x PBS (5 min, RT), to quench the formaldehyde and terminate the cross-linking reaction. The cells were washed twice with 1x PBS and permeabilised with 0.5% Triton X-100 in 1x PBS (5 min, RT). After two additional PBS washes, the coverslips were blocked with antibody dilution buffer (ADB: 1% BSA, 0.2% Cold Water Fish Skin Gelatin, 0.05% Triton X-100 in 1x PBS; 30 min, RT). Rabbit anti-Rad51 (7946 ²⁸, 1:1000 dilution) and mouse anti-γH2A.X (RRID:AB_309864, 05-636, Millipore, 1:500 dilution) primary antibodies were applied (2h, RT). After incubation with primary antibodies, the coverslips were washed twice with 0.05% Triton X-100 in 1x PBS (PBS-T) and once with ADB (5 min on an orbital shaker, RT). Following incubation (1h at RT) with anti-mouse Alexa Fluor 555 (RRID:AB_2535846, A-21425, Invitrogen, 1:400 dilution) and anti-rabbit Alexa Fluor 488 (RRID:AB_2534114, A-11070, Invitrogen, 1:400 dilution) secondary antibodies, the coverslips were washed three times with PBS-T (5 min on an orbital shaker, RT). The coverslips were finally air-dried at RT and mounted on glass slides with ProLong Gold antifade reagent with DAPI ({"type":"entrez-protein","attrs":{"text":"P36935","term_id":"549826","term_text":"P36935"}}P36935, Invitrogen).

Images acquired on an Olympus Fluoview FV1000 confocal laser-scanning microscope, using fixed parameters, were converted to RGB TIFF format using the Fiji (RRID:SCR_002285) distribution of ImageJ ²⁹. RAD51 and γH2AX nuclear foci were automatically quantified using the FoCo algorithm ³⁰. Noteworthy, filters were applied for the minimum radius of nuclei (15 pixels, blue), the minimum radius of foci (γH2A.X: 3 pixels, red; RAD51: 2 pixels, green) and the minimum intensity of foci (γH2A.X: 0.4, red; RAD51: 0.24, green). The number of foci of the first 180 cells scored was analysed using Prism 7 (RRID:SCR_002798, GraphPad Software).