γ-H2AX Assay

Immunofluorescent cytochemical staining for g-H2AX foci was performed. Cells were grown in chamber slides and exposed LB100 (2.5 mmol/L) for 4 hours prior to administration of 5Gy or sham radiation. Cells were fixed with 2% paraformaldehyde, washed with PBS, permeabilized with 1% Triton X-100, washed again with PBS, and blocked with 1% BSA. Mouse anti-g-H2AX antibody (Millipore) was added at 1:500 and incubated overnight at 4C. Cells were washed with 1% BSA and goat anti-mouse-FITC antibody (Jackson ImmunoResearch) was added at 1:100 and incubated 1 hour at room temperature. Nuclei were counterstained with DAPI (Sigma). Coverslips were mounted with VectaShield anti-fade solution (Vector Labs) and cells were examined on a Leica DMRXA fluorescent microscope (Leica Microsystems). γ-H2AX foci were quantitated in 50 cells per condition.