Rad51 Nuclear Foci Assay

780 cells were trypsinized 24 h after transfection and plated into 8-well chamber slides (1.5 × 10⁵ cells per well). Two slides were prepared, and cells were allowed to attach overnight. One slide was then subjected to IR and another was left untreated as the control slide. Cells were fixed and subjected to Rad51 staining 3 h after irradiation as previously described14 (link). A rabbit polyclonal anti-Rad51 (H-92, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the primary antibody, and FITC-conjugated goat anti-rabbit IgG (SouthernBiotech, Birmingham, AL, USA) was used as the secondary antibody. Slides were counter-stained with DAPI and mounted with antifade (Invitrogen, Carlsbad, CA, USA). Approximately 200 cells (ranging from 195 to 220 cells) were scored for each sample, and the percentages of cells with at least five Rad51 nuclear foci were calculated. Cell images were captured with a 100× objective.