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. 2008 Sep 4;36(17):5678–5694. doi: 10.1093/nar/gkn550

Figure 5.

Figure 5.

Comparison of DSB repair kinetics as measured by pulsed-field gel electrophoresis (PFGE) with the development of γ-H2AX foci. (A) Plateau-phase A549 cells were exposed to 20 Gy or 1 Gy X-rays and analyzed by PFGE (157) or γ-H2AX immunofluorescence (158), respectively, at the indicated time points. PFGE results (squares) have been normalized to the signal measured at 0 h, while the γ-H2AX results (circles) have been normalized to the maximum number of foci scored. The number of foci per cell was quantified using the Leica Q-Win software with the help of a special routine developed for foci counting. Foci were counted on 3D picture stacks generated on a Leica SP5 confocal microscope. γ-H2AX results were normalized to the maximum number of foci scored per cell—typically reached between 30 min and 1 h after IR. (B) Typical gel used to generate the PFGE results shown in A. DNA is stained with ethidium bromide. (C) Examples of γ-H2AX immunofluorescence at different times after exposure to IR (1 Gy). Cells were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with a phosphospecific primary anti-γ-H2AX antibody and an Alexa 568-labeled secondary antibody. Red shows γ-H2AX foci, blue nuclei stained with DAPI.