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Flash-comet assay
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Drug Safety & Toxicology)ORCID iD: 0000-0003-2471-1432
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Drug Safety & Toxicology)
2020 (English)In: MethodsX, ISSN 1258-780X, E-ISSN 2215-0161, Vol. 7, article id 101161Article in journal (Refereed) Published
Abstract [en]

In the present paper, we present a substantially revised protocol of the widely used SCGE assay performed under alkaline conditions. In our updated version of the comet assay, which we call the Flash-comet, LiOH is used instead of NaOH during the unwinding and electrophoresis. This allows a higher voltage during the electrophoresis (5 V/cm instead of 0.7 V/cm), making it possible to reduce the unwinding time from 20 to 40 to 2.5 min, and the electrophoresis time from 10 to 20 to 1 min. Still, the Flash-comet was found to detect DNA strand breaks and alkali-labile sites with a higher degree of sensitivity than the conventional protocol in cells that had been exposed to H2O2 or ionizing radiation. In order to prevent alkaline hydrolysis of DNA, the wash and lysis solutions have been modified in the Flash-comet protocol. By using an alkaline LiOH-based medium, the Flash-comet allows for much shorter times for both unwinding and electrophoresis than the conventional comet assay without compromising the sensitivity. The reduced run-times of the unwinding and electrophoresis steps in the Flash-comet should also reduce the risk of laboratory-induced alkaline hydrolysis of DNA when evaluating the potential DNA-damaging effects of different types of xenobiotics.

Place, publisher, year, edition, pages
ELSEVIER , 2020. Vol. 7, article id 101161
Keywords [en]
DNA-damage, Gel electrophoresis, Genotoxicity testing, Gentle lysis, In vitro comet assay, Low conductivity electrophoresis solution, Single cell analysis
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:uu:diva-434712DOI: 10.1016/j.mex.2020.101161ISI: 000607660800032PubMedID: 33304837OAI: oai:DiVA.org:uu-434712DiVA, id: diva2:1529812
Available from: 2021-02-19 Created: 2021-02-19 Last updated: 2021-02-19Bibliographically approved

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Bivehed, ErikHellman, Björn

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