Measuring DNA Damage in Irradiated Cells

RT112 cells were cultured on 10 mm No. 1 cover glasses (VWR) that had been sterilised with 70% ethanol and rinsed with PBS prior to ionising radiation. After incubation with bacterial supernatants and irradiation, cells were allowed to recover for indicated times prior to permeabilisation with 0.3% Triton X-100. Cells were fixed with ice cold 4% paraformaldehyde and blocked by incubation in 5% BSA, as described previousl y[84 (link)]. After incubation with primary γH2AX (mouse anti-phospho-Histone H2A.X (Ser139) IgG; clone JBW301, Millipore) and secondary (goat anti-mouse IgG, Alexa Fluor 488, ThermoFisher) antibodies, coverslips were mounted on microscopy slides using mounting reagent, Flouromount G, with DAPI (Invitrogen). Fluorescent foci were imaged using a Zeiss 710 confocal microscopy using either a 40X or 63X objective. All microscopy images were analysed with FIJI (ImageJ) software.