Flash-comet assay
| Autor: | Erik Bivehed, Björn Hellman |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Lysis
Flash-comet DNA damage Clinical Biochemistry Pharmacology and Toxicology 010501 environmental sciences 01 natural sciences Genotoxicity testing 03 medical and health sciences chemistry.chemical_compound Low conductivity electrophoresis solution lcsh:Science Alkaline hydrolysis 030304 developmental biology 0105 earth and related environmental sciences Gel electrophoresis 0303 health sciences Chromatography Single cell analysis Chemistry DNA-damage food and beverages Gentle lysis Method Article Farmakologi och toxikologi Comet assay Medical Laboratory Technology Electrophoresis In vitro comet assay lcsh:Q sense organs DNA |
| Zdroj: | MethodsX, Vol 7, Iss, Pp 101161-(2020) MethodsX |
| Popis: | 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. Graphical abstract Image, graphical abstract |
| Databáze: | OpenAIRE |
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