The Comet Assay: Automated Imaging Methods for Improved Analysis and Reproducibility

Abstract

Sources of variability in the comet assay include variations in the protocol used to process the cells, the microscope imaging system and the software used in the computerized analysis of the images. Here we focus on the effect of variations in the microscope imaging system and software analysis using fixed preparations of cells and a single cell processing protocol. To determine the effect of the microscope imaging and analysis on the measured percentage of damaged DNA (% DNA in tail), we used preparations of mammalian cells treated with etoposide or electrochemically induced DNA damage conditions and varied the settings of the automated microscope, camera, and commercial image analysis software. Manual image analysis revealed measurement variations in percent DNA in tail as high as 40% due to microscope focus, camera exposure time and the software image intensity threshold level. Automated image analysis reduced these variations as much as three-fold, but only within a narrow range of focus and exposure settings. The magnitude of variation, observed using both analysis methods, was highly dependent on the overall extent of DNA damage in the particular sample. Mitigating these sources of variability with optimal instrument settings facilitates an accurate evaluation of cell biological variability.

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair

“…For the chicken DT40 cells, the tail DNA percentage, reflecting the number of SSBs (% DNA in tail) [ 31 ], was measured for cells that had been exposed to 25 μM H 2 O 2 for 20 min on ice, and cells that had been exposed to 25 μM H 2 O 2 for 20 min on ice followed by a 30 min repair period at 39.5°C. For DT40 cells, a Comet Analysis System was used to quantify the comet tails (Komet 3.0, Kinetic Imaging Ltd., Liverpool, UK).…”

Meiosis-Specific Functions of Kinesin Motors in Cohesin Removal and Maintenance of Chromosome Integrity in Budding Yeast

“…5B). To quantify the defect, we compared the percentages of DNA in the head and tail parts of the comet among the test samples (see Materials and Methods) (56). A reduction in the percentage of DNA in the head region is accompanied by an increase in the percentage of DNA in the tails of H 2 O 2 -treated and cin8Δ kip3Δ spheroplasts (Fig.…”

Cellular Reference Materials for DNA Damage Using Electrochemical Oxidation

“…However, the inherent bio-variability of the cell's response to various steps of this procedure requires large numbers of cells to obtain a representative average. To obtain quality metrics, for most applications, about 100 cells are analyzed and this is practical only using an automated system for data collection and analysis [10,17]. Another source of variability inherent in the comet assay is its multistep experimental procedure that contributes variation during lysis, electrophoresis, staining and imaging steps [9].…”

“…Another source of variability inherent in the comet assay is its multistep experimental procedure that contributes variation during lysis, electrophoresis, staining and imaging steps [9]. In addition, there is no consensus as to which single parameter is the best representative of the DNA damage extent comets, based on previous measurements of the average imaging reproducibility [10]. Histograms of all three replicate measurements (separate cultures on electrodes) are given in Supplementary Figure S2.…”

“…e comet assay, however, has been known to suffer from significant experimental variability from lab to lab, traceable to changes in certain steps of the procedure [3,9]. In a previous study, we examined the role of the microscope imaging and analysis on the assay output variation [10]. Attempts have been made to reduce comet assay variation by adopting a completely standardized protocol but identification of one that is universally accepted using equivalent reagents and instrumentation remains to be established [11,12].…”