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DNA Damage and Repair - Quantification of Sub-Cellular Events Using Automated Confocal Imaging

owed very few foci next to cells containing a large number of foci ( Figure 1 ). We therefore sampled on average 120 cells/well in a 33 montage. Data was imported into BD Image Data Explorer where dose-response curves and Z data were generated ( Figures 3 and 4 ).

Discussion

The ability to acquire confocal, collapsed stack images has allowed us to develop an automated assay relying on identification of small sub-nuclear features, namely foci of DNA damage and repair. The improved statistical performance of the assay in confocal, collapsed stack mode suggests that this assay indeed can be applied to a high-content screening environment.

We noted that even untreated cells contained some foci, suggesting that under basal conditions, some DNA doublestrand breaks are present and repair occurs. Phosphorylated H2AX accumulation was seen immediately after chemical treatment confirming the rapid time course of the activation and recruitment of this protein to the sites of DNA damage.
The subsequent accumulation of 53BP1 foci peaked between 30 and 90 minutes and is in agreement with published reports (Schultz et al . 2000). To our knowledge, the EC50 for H2O2-induced H2AX and 53BP1 foci formation has not been published and we believe that our assay can aid the characterization of other chemical agents and DNA repair proteins in a similar manner. The appearance of the 53BP1 foci in the same spatial location after 30 minutes confirms reports of rapid activation of the repair signaling cascade (Schultz et al . 2000)

Although we did not investigate the signaling properties between these two proteins, the assay is very suitable to investigate whether intermediate molecules are involved or whether 53BP1 is recruited by activated H2AX directly.

The comparatively straight-forward a
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