Quantifying DNA Damage Response

Cells were transfected with the indicated siRNAs and were trypsinized 24 h after transfection, seeded on glass coverslips in a 24-well plate for 24 h, and irradiated at the indicated doses. γ-H2AX immunofluorescence analysis was carried out as described previously [24 (link)]. At 0 h, 0.5 h, 6 h, 12 h, and 24 h post irradiation, cells were fixed with 4 % paraformaldehyde (15 min, AppliChem, Darmstadt, Germany) at room temperature (RT) and permeabilized by the addition of 0.25 % Triton-X 100 in PBS for 15 min, followed by blocking in 5 % bovine serum albumin (BSA) in PBS for 30 min. Next, cells were incubated with Phospho-Histone H2AX (Ser139) (20E3) rabbit mAb (1:1000; Cell Signal Technology; #9718S), followed by the appropriate Alexa 488-conjugated (green; Molecular Probes) secondary antibodies Subsequently, nuclei were counterstained with DAPI solution (Invitrogen) and coverslips were mounted with Vectashield (Vector Laboratories, Peterborough, UK). Images were taken using an Olympus confocal imaging system (Olympus FV100) for the quantification of γH2AX foci formation. At least three independent experiments were performed for each data point.