Miniaturizing the comet assay with 3D vertical comets

Abstract

BackgroundThe 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.MethodsA 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.ResultsThree‐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.ConclusionsVertical 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. Cytometry Part A 51A:26–34, 2003. © 2002 Wiley‐Liss, Inc.

A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks

“…Since the introduction of the comet assay, many modifications have been introduced to increase its performance [ 9 ] and enhance throughput [ 10 - 12 ]. A method using fluorescence analysis of DNA unwinding to measure DNA damage was also modified to decrease processing time [ 3 , 6 ].…”

Heterogeneity in DNA damage using the comet assay

“…Automated comet analysis systems (54) have been developed that could overcome this limitation provided they do not introduce additional heterogeneity (e.g., by including overlapping comets that are counted as one). A novel confocal imaging approach using vertical comets can also operate automatically to sample 6 comets/min; however, additional time is required for image processing (55). A more challenging and interesting issue is that, once heterogeneity in response is detected, it will necessitate a consideration of the basis for that heterogeneity.…”

The automated FADU-assay, a potential high-throughput in vitro method for early screening of DNA breakage

“…by higher throughput. Several modifications of the comet assay have been reported over the past years (Kiskinis et al, 2002;Baert and Van Oostveldt, 2003;Morley et al, 2006;Cordelli et al, 2007;Wooda et al, 2010), however, its automation has only been successful in some of the experimental steps (Frieauff et al, 2001;Wooda et al, 2010).…”