2.8. HRR Function by Immunofluorescence Based γH2AX-RAD51 Assay

HRR was assessed by immunofluorescence microscopy, as previously described [15]. Cells were treated for 48 h with 10 μM rucaparib alongside vehicle alone controls (0.5% DMSO). To quantify DNA damage, cells were stained with mouse monoclonal anti-phospho-histone H2A.X (Ser139) antibody (Upstate/Millipore, Burlington, NJ, USA) at 1:1000 and rabbit monoclonal anti-RAD51 antibody (AbCam, Cambridge, UK) at 1:500 to assess downstream HRR functional repair. Secondary antibodies used were Alexa 488 conjugated goat anti-rabbit and Alexa 546 conjugated goat-anti mouse (Invitrogen, Waltham, USA), both at 1:1000. The nuclei were stained with DAPI. γH2AX and RAD51 foci in each cell were quantified using ImageJ software and data analysed using GraphPad Prism. A >2-fold increase in γH2AX foci formation was used as an indicator of DNA damage induction following rucaparib treatment, and a >2-fold increase in RAD51 foci formation was indicative of functional HRR. The same method was also used for analysing the HRR ability of primary cultures with minor modifications.