Alexa 546 conjugated goat anti mouse

Cells were treated for 24 h with control vehicle (DMSO) and 1 μM VE-821 with or without 10 μM olaparib. Cells were stained with mouse monoclonal anti phospho-Histone H2A.X (Ser139) antibody (SCBT) at 1:500 and rabbit monoclonal anti RAD51 antibody (CST: 8875S) at 1:250. Secondary antibodies used were Alexa 488 conjugated goat anti rabbit and Alexa 546 conjugated goat anti mouse (Invitrogen, Thermo Fisher Scientific), both at 1:1000. Cells were imaged using a Leica DM6 microscope and Leica Application Suite (LAS) X software (Leica Microsystems, Wetzlar, Germany). The number of RAD51 foci in each cell and total nuclear fluorescence intensity for γH2AX were quantified using ImageJ software (Version 1.52p; Java 1.8.0_172 (64-bit)) and data was plotted using GraphPad Prism v6 (San Diego, CA, USA).

HRR was assessed by immunofluorescence microscopy, as previously described [15 (link)]. 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.