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.