Miniaturizing the comet assay with 3D vertical comets.
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.
Baert
Philippe
P
Department of Molecular, Biotechnology, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Belgium.
Van Oostveldt
Patrick
P
D000465:Algorithms; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000648:ultrastructure*; D020552:Comet Assay / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis*; D004249:DNA Damage / Q000235:genetics*; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D021621:Imaging, Three-Dimensional; D051379:Mice; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012984:Software
D004247:DNA