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.
Highlights
The comet assay offers sensitive detection of both single and double stranded breaks
At least three replicates were produced at each treatment
Establishing the effect of imaging parameters will be critical in future examination of the individual steps involved in the comet assay procedure[8,9]
Summary
The comet assay offers sensitive detection of both single and double stranded breaks. The inherent heterogeneity of the cells used in the comet analysis requires that a statistically relevant number of cells must be examined to obtain a representative average. This becomes practical only using an automated system for data collection and analysis[10]. Another source of variability is related to the imaging and analysis procedures required to quantify the results. In this study we focus on the sources of variability in the imaging and analysis of comet assay samples using public domain and commercially available software. We demonstrate the utility of automated image capture to increase the number of comet images and improve statistics, following etoposide and electrochemical oxidative treatments of the cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
