中性单细胞凝胶电泳分析

Elisa  Boutet-Robinet; Didier  Trouche; Yvan Canitrot

doi:10.21769/BioProtoc.915

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Peer-reviewed

Neutral Comet Assay

中性单细胞凝胶电泳分析

Elisa Boutet-Robinet email

Didier Trouche

Yvan Canitrot

发布: 2013年09月20日第3卷第18期 DOI: 10.21769/BioProtoc.915 浏览次数: 37580

评审: Lin FangFanglian He

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The authors used this protocol in:

Cover of The Journal of Cell Biology, featuring study using the protocol.

Dec 2012

实验方案合集

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Abstract

The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The following document describes the protocol to realize a neutral comet assay. This assay can be applied to different cell types and has been useful for numerous applications in fields of toxicology or DNA damage and repair.

Keywords: Comet assay (彗星实验)

Genotoxicity (genotoxicity)

DNA damage (DNA损伤)

Materials and Reagents

Cells to analyze
Low Melting Point (LMP) Agarose (Sigma-Aldrich, catalog number: A9414 )
Seakem^® Agarose (Ozyme, catalog number: LON50004 )
PBS (Ca²⁺ and Mg²⁺-free phosphate-buffered saline)
5 N NaOH
0.5 M EDTA disodium salt solution (pH 8)
Trisma base
Triton X-100
N-Lauroylsarcosine (Sigma-Aldrich, catalog number: L5125 )
Dimethylsulphoxide (DMSO)
Absolute ethanol
Ethidium bromide (10 mg/ml)
Trypsin/EDTA
Sodium acetate
Lysis solution (see Recipes)
Electrophoresis solution (see Recipes)

Equipment

Microscope Super Frost plus glass slides
Malassez chamber
Microscope coverslips (22 x 22 mm)
Microscope coverslips (24 x 32 mm)
Centrifuges
Electrophoresis tank: Econo-Submarine (20 cm x 30 cm) (C.B.S. Scientific, USA)
Fluorescence microscope, camera and software (e.g. Nikon Eclipse 50i microscope equipped with a Luca S camera and Komet 6.0 software)

Software

Komet 6.0 software (Andor Technology)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Boutet-Robinet, E., Trouche, D. and Canitrot, Y. (2013). Neutral Comet Assay. Bio-protocol 3(18): e915. DOI: 10.21769/BioProtoc.915.
Courilleau, C., Chailleux, C., Jauneau, A., Grimal, F., Briois, S., Boutet-Robinet, E., Boudsocq, F., Trouche, D. and Canitrot, Y. (2012). The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks. J Cell Biol 199(7): 1067-1081.