Single-cell gel electrophoresis assay monitors precise kinetics of DNA fragmentation induced during programmed cell death

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Sudhir Chandna

Abstract

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. 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. In contrast to results of previously published studies, comets with different shapes reflecting progressive stages of DNA fragmentation were observed. Of these...Continue Reading

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Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis

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