Quantifying DNA Damage Foci in Irradiated Lymphocytes

Following irradiation, the separated lymphocyte samples were incubated at 37 °C in a 5% CO₂ humidified atmosphere to simulate in vivo repair. The 0.5 Gy samples were held for 0.5 h and the 4 Gy specimens for 24 h, to assess the initial and residual radiation-induced foci, respectively. Samples were then processed for the assessment of gamma-H2AX foci using a standard technique [22 (link)]. In brief, the lymphocytes were spotted onto adhesive slides, fixed with formaldehyde (Polysciences Inc., Warrington, PA, USA), permeabilised with Triton X (Sigma-Aldrich, Dorset, UK), blocked with bovine serum albumin (Fisher Scientific, Loughborough, UK) and immunostained for gamma-H2AX and 53BP1 using fluorophore-conjugated secondary antibodies (AlexaFluor 488 goat anti-mouse/AlexaFluor 555 goat anti-rabbit, Invitrogen, Paisley, UK). Fifty cells per sample were scored manually by one person, (with the exception of 8 donors), for gamma-H2AX and 53BP1 foci, using a Nikon Optiphot 2 fluorescence microscope equipped with separate filters to visualise 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothio-cyanate (FITC) and Texas Red. Only gamma-H2AX foci co-localised with 53BP1 were scored, as this reduces the possibility of erroneously scoring fluorescent antibody aggregates as foci [23 (link)].