IMAGE-ANALYSIS OF COMET ASSAY MEASUREMENTS

In the last decade the 'comet assay' or 'single cell gel electrophores is assay' has been established as a sensitive method for the detection of DNA damage and the measurement of its recovery. The results publis hed in the literature have often been obtained with different methods for comet structure measurement. In most cases these data are not comp arable with each other. Even when using similar systems for the analys is, it is difficult to obtain matching data. This presentation will de scribe some technical aspects of our measurement equipment and evaluat ion software. It focuses on necessary experimental conditions to minim ize errors in obtaining such data. The software developed here allows the rapid analysis of the microscopic samples (<2 a per image). The im age analysis was designed with respect to the morphological shapes of comet cells, which were investigated with a confocal laser microscope. The system is built with standard components which are commercially a vailable. As a measure of the amount of DNA damage the ratio of fluore scence intensity was used inside the comet tail and the fluorescence i ntensity of the comet head. Other parameters such as DNA content, come t area, head radius, tail length and tail moment are also determined. The reproducibility of the system has been evaluated in several experi ments over a period of 5 years.