The Comet Assay: Automated Imaging Methods for Improved Analysis and Reproducibility
| Autor: | Signe Braafladt, Donald H. Atha, Vytas Reipa |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Reproducibility Multidisciplinary Microscope Focus (geometry) DNA damage business.industry Reproducibility of Results Biology Article law.invention Comet assay 03 medical and health sciences 030104 developmental biology Software law Image Processing Computer-Assisted Humans Comet Assay Image analysis Software analysis pattern business Biomedical engineering |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/srep32162 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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