High throughput Comet assay using 96-well plates

The single-cell gel electrophoresis or Comet assay is becoming established as an industrial genotoxicity screening test. The aim of this study was to increase the throughput of compounds tested and to minimize the amount of test compound needed for an assay. We modified practical aspects of our standard protocol and designed an experimental procedure suitable for use with 96-well plates. By using a suspension culture rather than attached cells, the modified protocol enabled parallel testing of four compounds on a single microplate (10 duplicate concentrations per compound). A significant reduction in work time was achieved by replacing the previously used Trypan blue dye exclusion (TBDE) test by an automated measurement of ATP levels as the concurrent viability test. The rapid and easy to perform ATP test was carried out towards the end of the 3 h treatment. In this way we were able to select for further analysis and slide preparation only those concentrations which induced the desired range of cytotoxicity. The suitability of the modified test conditions and reproducibility of test results was demonstrated by results obtained with standard mutagens and eight drug candidates tested at various concentrations. In each case the results obtained with the standard and the modified protocols were comparable. By introducing the changes to our standard protocol, combined with automated image analysis, we were able to more than double our previous throughput.