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The alkaline comet assay, in vivo and in vitro, is currently used in several areas of research and in regulatory genotoxicity testing. Several efforts have been made in order to decrease the interexperimental and inter-laboratory variability and increase the reliability of the assay. In this regard, lysis conditions are considered as one of the critical variables and need to be further studied. Here, we tested different times of lysis (from no lysis to 1 week) and two different lysis solutions in human lymphoblast (TK6) cells unexposed or exposed to X-rays. Similar % tail DNA values were obtained independently of the time of lysis employed for every X-ray dose tested and both lysis solutions. These results, taken together with our previous ones with methyl methanesulfonate and H22, which showed clear lysis-time dependence, support that the influence of the lysis time in the comet assay results depends on the type of lesion being detected; some DNA lesions may spontaneously give rise to apurinic or apyrimidinic (AP) sites during the lysis period, which can be converted into strand breaks detectable with the comet assay. Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced. However, the same lysis conditions (i.e. lysis time and lysis solution) should be used when comparing results between different experiments or laboratories. [ABSTRACT FROM AUTHOR]

The single-cell gel electrophoresis—the comet assay—has proved to be a sensitive and relatively simple method that is much used in research for the analysis of specific types of DNA damage, and its use in genotoxicity testing is increasing. The efficiency of the comet assay, in terms of number of samples processed per experiment, has been rather poor, and both research and toxicological testing should profit from an increased throughput. We have designed and validated a format involving 96 agarose minigels supported by a hydrophilic polyester film. Using simple technology, hundreds of samples may be processed in one experiment by one person, with less time needed for processing, less use of chemicals and requiring fewer cells per sample. Controlled electrophoresis, including circulation of the electrophoresis solution, improves the homogeneity between replicate samples in the 96-minigel format. The high-throughput method described in this paper should greatly increase the overall capacity, versatility and robustness of the comet assay. [ABSTRACT FROM AUTHOR]