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Background: Single-cell gel electrophoresis, or the comet assay, a technique widely used for DNA damage analysis, has been used recently for detecting DNA fragmentation in cells undergoing apoptosis. However, the number of variants of this assay used thus far primarily detected the late stages of DNA fragmentation. Therefore, monitoring the progression of DNA fragmentation, which could greatly improve the analysis of cell death induction and progression at the single-cell level, has not been possible with this assay., Methods: In the present study, a modification of the original neutral comet assay developed by Ostling and Johanson (Biochem Biophys Res Commun 123:291-298, 1984) was used to detect various stages of DNA fragmentation. This assay involves cell lysis with anionic detergents at nearly neutral pH (9.5) and does not include high salt concentration, unlike most other published methods. BMG-1 human glioma cells were induced to undergo programmed cell death by treating with a large dose (100 microM) of etoposide, and comets were prepared after different durations (1-24 h) of treatment., Results: In contrast to results of previously published studies, comets with different shapes reflecting progressive stages of DNA fragmentation were observed. Of these, six distinct shapes were identified and divided into three different categories based on the extent of fragmentation. Type A comets had a large head separated by a narrow "neck" region from an oval bulging tail that indicated initiation of fragmentation. Type B and C comets had a constantly diminishing head associated with a corresponding expansion of the tail and reflected intermediate and late stages of fragmentation, respectively. Type A and B comets appeared at a high frequency during early time points (1-6 h), whereas type C comets that indicated late stages of fragmentation were observed only after extended treatment (24 h). As a result, an elaborate kinetics of the progression of DNA fragmentation could be obtained., Conclusion: The present single-cell gel electrophoresis assay offers a significant improvement in monitoring the kinetics of DNA fragmentation induced during programmed cell death. Coupled with its simplicity and the ability to detect responses of small cell subpopulations, this method can be used for a reliable and sensitive analysis of the progression of cell death in different cell types and treatment conditions., (Copyright 2004 Wiley-Liss, Inc.)

Background: The comet or single-cell gel electrophoresis assay is a sensitive method for the detection of DNA damage. The main drawback of comet sampling is the low cell density necessary to prevent nucleus overlap after electrophoresis, which limits large-scale high throughput screening. Another problem may be inconsistent comet focusing. We investigated whether an approach based on three-dimensional (3D) confocal microscopy might be beneficial for these concerns., Methods: A vertical comet assay enabling three-dimensional confocal comet imaging of nuclei seeded at very high density was developed together with dedicated software algorithms to retrieve quantitative data at the single cell level., Results: Three-dimensional confocal comet imaging greatly relieved the user interactions of our nonautomated two-dimensional comet sampling procedure. Batches of comets were blindly sampled, and confocal sectioning improved the clarity of the images and the accuracy of comet sampling. A 1-Gy dose response was readily established. The sampling speed was competitive with that of commercial packages., Conclusions: Vertical comet imaging is a new concept for fast and user-friendly comet sampling that allows miniaturization of the assay. It may become an essential step toward high throughput screening and exploit the benefits of confocal imaging., (Copyright 2002 Wiley-Liss, Inc.)