The comet assay was first developed by ostling and johanson in 1984 by demonstrating the migration of dna fragments from nuclei under a neutral condition 1. Excellent scholarly papers with concise descriptions of statistical analysis and recommendations for tests have been published lovell et al. Complete this form with the information that we need in order to set up your screening. I dont use any slide coating to perform the comet assay. If you are looking to start the comet assay in your laboratory, then you will be reassured to know that the assay does not require any special equipment. Our oxiselect 96well comet assay kits provide a higherthroughput way to screen for general dna damage, regardless of the source or nature of the damage. Introduction single cell gel electrophoresis scge or the comet assay is a versatile, sensitive yet simple and economical technique used to measure dna damage and repair in individual cells. Background trevigen s cometassay, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating dna damage in cells. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress. What is the treatment time and compound concentrations. The comet assay or single cell gel electrophoresis assay permits the quantification of dna damage in individual cells based on the migration of dna structures in an electric. Differences in quantification of dna doublestrand breaks assessed by 53bp1 h2ax focus formation assays and the comet assay in mammalian cells treated with irradiation and nacetyllcysteine tomomi kurashige, mika shimamura, and yuji nagayama. Its simplicity, sensitivity, speed and economy make it a prime choice in genotoxicity testing, ecogenotoxicity, human epidemiology studies as well as mechanistic studies of dna damage and repair.
The methods used are mostly based on a commercially available comet assay kit from trevigen inc. The principle of the assay is based upon the ability of denatured, cleaved dna fragments to migrate out of the nucleoid under the influence of an electric field, whereas. The increasing trend in damage is accurately measured by opencomet. The mechanism of formation of comets under neutral or alkaline conditions is best understood by analogy with nucleoids, in which relaxation of dna supercoiling in a structural loop of dna by a single dna break releases that loop to extend into a haloor, in the case of the. The in vivo alkaline comet single cell gel electrophoresis assay hereafter called simply the comet assay is used for the detection of dna strand breaks in cells or. Following gentle cell lysis, and for the alkaline cometassay, samples are treated with alkali to unwind and denature the dna and hydrolyze sites of damage. Development of the in vivo alkaline comet assay for use with spraguedawley rat skin f.
Ap biology 2017 frq 6 student samples college board. The single cell gel electrophoresis scge or comet assay is a sensitive and rapid technique for quantifying dna damage and repair from in vivo and in vitro samples of eukaryotic and plant cells. Such variation sometimes makes it difficult to compare results between laboratories, and even within the same lab. Comet assay kit 3well slides ab238544 is a fast and sensitive kit for the measurement of cellular dna damage. Excellent scholarly papers with concise descriptions of sta. Cultrex cell migration assay 96 samples 95036838 trevigens cultrex. If treatment is being performed by trevigen cell assays what type of cells are required. December 20 1 draft oecd guideline for the testing of chemicals in vivo mammalian alkaline comet assay introduction 1. Follow the instructions to install and launch opencomet.
We often come across publications that unfortunately have not taken advantage of statistical models in design and analysis of comet assay results. The nucleus of an individual cell is placed on a microscope slide coated with an agarose gel. The comet assay, also known as singlecell gel electrophoresis scge, has become an attractive assay in the assessment of dna damage. It is a convenient way to screen for general dna damage, regardless of the source or nature of the damage. Hydrophobic barrier allows easy application of cells directly to slide, and treatment with any of several trevigen dna repair enzymes. In addition, the recommended exposure regimen described in this guideline is not optimal and longer exposures or sampling times would be necessary for a meaningful analysis of dna strand breaks in mature sperm. Under alkaline conditions ph, the comet assay can detect single and double stranded breaks, resulting, for example, from direct interactions with dna, alkali labile sites or as a consequence of transient dna strand breaks resulting from dna excision repair. The cometassay, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating dna damage. In comet assays, cells are immobilized in a bed of low melting point agarose, on a trevigen cometslide. Trevigen s comet analysis software provides the computational power to efficiently analyze digital images presenting fields of cells run in the comet assay.
Trevigens cultrex endothelial cell invasion assay accelerates the screening process for compounds that influence vascular endothelial cell invasion through the basement membrane. For both assay types, cells are lysed and the nucleoids subjected to electrophoresis. The principle of the assay is based upon the ability of denatured, cleaved dna fragments to. The neutral assay performed on trevigen neutral comet assay control cells with different levels of dna damage. The mechanism of formation of comets under neutral or alkaline conditions is best understood by analogy with nucleoids, in which relaxation of dna supercoiling in a structural loop of dna by a single dna break releases that loop to extend into a haloor, in the case of. Essentially, anything that can cause dna damage or denaturation except the factors being researched is to be avoided. An electric current is applied to the gel that causes dna to move electrophoresis, and. The standard alkaline comet assay was performed using alkaline cometassay control cells cc, treated with. In order to visualise your cells, you will need a microscope upright or inverted style with fluorescence.
Trevigen s cometassay silver kit provides all the reagents for silver staining of the processed cometslide allowing visualization by standard light microscopy and providing permanent staining for sample archiving. The principle of the assay is based upon the ability of denatured cleaved dna fragments to migrate out of the cell under the influence of an electric potential, whereas undamaged supercoiled dna remains within the confines of the cell membrane when a current is applied. The comet assay helps to measure the single double strand dna breaks, alkali labile sites apurinic apyrimidinic sites, dna crosslinks. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. Cometslides allow direct application of comet lmagarose without base layers or pretreatment.
The use of trevigens cometslide shortens assay time and allows the rapid and reliable analysis of large numbers of samples. The comet assay is a versatile and sensitive method for measuring single and doublestrand breaks in dna. It is used in cancer research, in genotoxicity studies on environmental mutagens, and for screening compounds for cancer therapeutics. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. Trevigen announces the first standardized comet assay electrophoresis system es for the direct detection of dna damage the comet assay is the only direct method for the detection of dna damage in cells. Trevigens cometassay provides a simple and effective method for evaluating dna damage in cells. Administration of the treatment consists of daily doses over duration of 2 days or more. Comet assay as an indirect measure of systemic oxidative. We validated opencomet on neutral comet assay images, which present different characteristics from alkaline comet assay images. The comet assay, also called single cell gel electrophoresis scge, is a sensitive and rapid technique for quantifying and analyzing dna damage in individual cells. Simply embed cells in low melting agarose on a pretreated slide, y s eu nd r alk ic o t, p hdna f m g nuclear region i. Since then, the neutral comet assay is mostly used to detect doublestranded. The use of trevigen s cometslide shortens assay time and allows the rapid and reliable analysis of large numbers of samples.
The comet assay has traditionally been used due to its high sensitivity and simplicity, but comet scoring is a laborious. In comet assays, cells are immobilized in a bed of low melting point agarose. Hydrogen peroxide o neutral comet 40 30 20 10 to tl t2 h202 treatment. Standard alkaline comet assay slides were prepared as follows or obtained directly from trevigen.
I would not recommend adding additional agarose on top. Cometassay is the traditional single cell gel electrophoresis protocol to quantitate dna damage by immobilizing a mixture of low melt agarose and cells onto a treated glass slide. We present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. Trevigen s new comet assay kit, dashtm diffusion apoptosis slide h alo, wsd icr m n t fh ey g ju hours. Catalognum product name quantity price supplier category. The head is composed of intact dna, while the tail consists of singlestrand or doublestrand dna breaks.
Development of the in vivo alkaline comet assay for use. The product accompanying this document is intended trevigen. Opencomet is a plugin for the image processing program imagej. Comet assay to detect doublestranded breaks in dna a comet assay is a technique used to determine the amount of doublestrand breaks in dna dna damage in cells. The purpose of the comet assay is to identify substances that cause dna damage. Reagent kit for single cell gel electrophoresis assay catalog. All incubations were performed in duplicate and at.
The resulting image that is obtained resembles a comet with a distinct head and tail. The software applies state of the art image processing methods to automatically locate scorable. Comet assay measures dna breaks, induced by different factors. Cometassay, cometslide, rec and flare are trademarks of trevigen, inc. The comet assay is a sensitive technique for quantifying dna damage and repair. Opencomet can analyze a batch of comet assay images, identify the comets and measure their parameters. Bleomycin treatment t3 1 800 trevigen bleomycin healthy e 30 10 bleomycin alkaline comet hydrogen peroxide healthy increase in damage seen with increasing concentrations of bleomycin and hydrogen peroxide trevigen, inc. Single cell gel electrophoresis scge or the comet assay is a versatile, sensitive yet simple and economical technique. Comet assay single cell gel electrophoresis sigmaaldrich. Cometassay control cells for single cell gel electrophoresis. Since variability has been observed when performing the comet assay, trevigen has developed a cometassay electrophoresis system es figure 1 optimized for use with trevigens cometassay kits e. The standard alkaline comet assay procedure was performed using alkaline cometassay control cells cc, treated with increasing, proprietary.
Also, this chapter discusses how specifics of the protocols influence the conclusions about types of dna damage and what the limitations of this method are for detecting different types of dna damage. The comet assay is a single cell gel electrophoresis assay scge for simple evaluation of cellular dna damage. A cytogenetic study of new zealand nuclear test veterans. Jun 27, 2006 we present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. In a typical comet assay, electrophoresismethods and differences in cell preparations create a significant source of variation in comet tail parameters. The comet assay is a wellpublished method for measuring cellular dna damage. Combining a novel comet assay procedure with automated. In vivo mammalian alkaline comet assay introduction.
The in vivo alkaline single cell gel electrophoresis assay, also called alkaline comet assay is a method measuring dna strand breaks in eukaryotic cells. In order to visualise your cells, you will need a microscope upright. Trevigen, cometassay and cometchip are registered trademarks of. Reagent kit for single cell gel electrophoresis assay. If all factors except oxidative stress causing dna damage are kept constant, the amount of dna damage is a good indirect parameter of oxidative stress. Cell invasion products cell invasion cell migration cell adhesion 3d culture endothelial cell invasion tube formation angiogenesis. Essentially, anything that can cause dna damage or denaturation.
Trevigens comet analysis software provides the computational power to efficiently analyze digital images presenting fields of cells run in the comet assay. The comet assay is a sensitive and rapid method for dna strand break detection in individual cells, and the year 2009 represents the 25th anniversary of the first description of this methodology. The comet assay is an extremely sensitive dna damage assay. Evaluation of the dna comet tail shape and migration. Evaluating in vitro dna damage using comet assay protocol. The trevigen team is here to assist you with your research needs. Anova, comet assay, experimental design, nonparametric test, statistical analysis the comet assay can distinguish small differences in dna damage between differentsamplesofcells,implyingthatstatistical tests are important to assess whether this occurs by chance. In the alkaline cometassay, samples are treated with alkali to unwind and denature the dna and hydrolyze sites of damage prior to electrophoresis. Detection of deoxyribonucleic acid dna damage at the level of an individual eukaryotic cell warrants high significance in the fields of toxicology, pharmaceuticals, genotoxicity testing, environmental human biomonitoring, diagnosis of genetic disorders etc. The comet assay, or single cell gel electrophoresis assay, permits the.
Each treated group is composed of a minimum of 5 animals of one sex or of each sex as appropriate. Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit. Such variation sometimes makes it difficult to compare results. Development of the in vivo alkaline comet assay for use with. Trevigens cometassay, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating dna damage in cells. A positive and a vehicle control group are also used. We use the specialized slides from trevigen that are made for performing the assay. Trevigens cometassay, or single cell gel electrophoresis assay, provides a simple and.
Kits contain comet slides, reagents, and a fluorescent dye to visualize cells under an epifluorescence microscope. Formation of dna damage was assayed by the alkaline comet assay essentially as. Automated imaging and analysis of a novel comet assay to. The principle of the assay is based upon the ability of denatured cleaved dna fragments to migrate out of the cell under the influence of an electric potential the comet tail, whereas undamaged supercoiled dna remains within the confines of the cell membrane the.
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