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UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS Guest
2024-11-22 2:11 |
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Conference: Bucharest University Faculty of Physics 2012 Meeting
Section: Biophysics; Medical Physics
Title:
Use of single cell gel electrophoresis/comet assay for detecting DNA damage of L-929 cell lines
Authors:
Lucia Elena ENCIU (1), Diana SAVU (2), Doina GAZDARU (1)
Affiliation:
(1)Department of Biophysics,Faculty of Physics,University of Bucharest,405 Atomistilor,Magurele-Ilfov,Romania
(2)Horia Hulubei National Institute for Research and Development in Physics and Nuclear Engineering,Magurele-Ilfov,Romania
E-mail
enciu_lucia@yahoo.com
Keywords:
Comet assay, single cell gel electrophoresis, DNA damage
Abstract:
Optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH >13) version of the assay development by Singh et al. The pH >13 version is capable of detecting DNA single strand breaks (SSB), alkali-labile sites, DNA-DNA/DNA; protein cross liking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, its flexibility, ease of application, low cost, and a short time needed to complete a study.We used a method for measuring DNA damage of L-929 cell lines, based on the technique of electrophoresis. Cells embedded in agarose are lysed, subjected to an electric field, stained with a fluorescent DNA binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a "comet" with a brightly fluorescent head and tail region which increases as damage increases. We have used video image analysis to define appropriate features of the comet as a measure of DNA damage. The assay was optimized for lysing solution, lysing time, electrophoresis time of the cell lines. We irradiated the cells with UV, at different doses. The DNA migration was determined by its staining with ethidium bromide and by measuring the intensity of fluorescence. The "tail moment", the product of the amount of DNA in the tail and the main distance of migration in the tail, was the most informative feature of the comet image. Relative to other genotoxicity tests, the advantages of the single cell gel assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small number of cells per sample and flexibility.
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