( 1034) for 53BP1 respectively. The DNA ...)DNA Damage and Repair - Quantification of Sub-Cellular Events Using Automated Confocal Imaging

1034) for 53BP1, respectively. The DNA intercalating dye Hoechst 33342 (Invitrogen, H3570) was added at this point at 2 μg/mL and removed with the secondary antibodies after 1h at room temperature (light protected). Cells were washed as described above and wells replenished with 200 μL of PBS. Plates were sealed with adhesive foil and imaged on the BD Pathway Bioimager. Analysis of the image data was performed using BD IPLab for Pathway (Cat. No. 340989) and plotted using BD Image Data Explorer (Cat. No. 341039).

Results

Treatment of HT1080 cells with H₂O₂ rapidly induced the formation of phosphorylated H2AX and 53BP1 foci (Figure 1 ). There is a marked increase in the number and intensity of foci in H202 treated cells compared to control cells. The two foci shared a high degree of co-localization within nuclei as observed in the merged images.

Preliminary results had shown that the nuclear foci were not evenly distributed in the same focal plane ( Figure 2 ). We used the ability of the BD Pathway Bioimager to acquire 9 sections separated by 1.5 μm steps in confocal mode. To achieve the required spatial resolution, we used a 40 (NA 0.90) objective. Using the novel collapsed-stack acquisition feature, images were then automatically projected into a single focal plane with all objects appearing in focus.

This allowed segmentation and identification of objects within the nucleus using a customized image analysis routine written in BD IPLab for Pathway. The collapsed stack images were compared to images acquired in non-confocal mode ( Figure 2 shows part of an image containing a single nucleus). The analysis tool generated specific parameters such as foci count, foci area as percent nuclear area, average foci intensity and average foci size. On the cellular level, the response appeared highly heterogeneous and cells could be found that sh
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