Immerse the slide for 5 mins, and then repeat once more. Recommendations for conducting the in vivo alkaline comet assay. Is the comet assay a sensitive procedure for detecting. The nucleus of an individual cell is placed on a microscope slide coated with an agarose gel. Do not pour solutions directly over slides or drop slides i. The principle of the assay is based upon the ability of denatured, cleaved dna fragments to migrate out of the nucleoid under the influence. This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess dna damage. Comet assay iv has the ability to score comets from a live video. The in vivo comet assay was negative and it was concluded that formation of a genotoxic metabolite was unlikely.
The alkaline comet assay has many applications in genetic toxicology and human biomonitoring. The comet assay is a single cell gel electrophoresis assay scge for simple evaluation of cellular dna damage. The efforts to develop a standard procedure for the in vivo comet assay in genetic toxicology occurred concurrently with validations of the comet assay in biomonitoring studies. As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. Comet assay as an indirect measure of systemic oxidative. 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. Alkaline comet assay for assessing dna damage in individual. Feb 24, 2015 this apoptosis assay protocol explains comet assay protocol to determine apoptosis. Single cell gel electrophoresiscomet assay youtube. The comet assay is an efficient method to detect dna damage including single and doublestranded dna breaks. In two cases, an in vivo comet assay was performed although the standard genotoxicity tests were negative.
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. Experiences with the in vivo and in vitro comet assay in. Dec 19, 2017 a full explanation about bradford assay, coomassie brilliant blue and the calibration curve duration. The in vitro comet assay can be performed in any rodent or human cancer cell lines and human lymphocytes. What if you could score a comet slide in two minutes. Comet assay does not require any special apparatus or chemicals. Dec 19, 2017 for the love of physics walter lewin may 16, 2011 duration. 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. The comet assay or single cell gel electrophoresis scge is a technique to easily and rapidly detect and quantitate dna fragmentation. Nov 16, 2017 comet assay protocol natobioradproject natobioradproject. You can also send your messagequeries to the entire group.
View cometassay single cell gel electrophoresis assay 4250050k. The assay can detect dna double and singlestrand breaks, alkalilabile lesions, and singlestrand breaks associated with incomplete dna repair. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. Recommendations for conducting the in vivo alkaline comet. Ive performed a comet assay multiple times, improving on the procedure each time and remaking my solutions, but continue to get the same inconclusive results. Applicable for the analysis of either single or doublestrand dna breaks. The comet assay permits the quantification of dna damage and repair in single cell preparations and is applicable to any eukaryotic cell.
Contrary to the development of test procedures for the in vivo comet assay in animal models, the efforts to validate the assay in biomonitoring studies have also. Jurkat cells untreated left and treated right with etoposide before performing oxiselect comet assay under alkaline electrophoresis conditions at 33v 300 ma for 15 minutes. For the comet assay we used the standard procedure originally described by singh et al. This technique uses cells that are embedded into agarose, placed on a slide, which is then immersed in lysis solution. The in vivo comet assay single cell gel electrophoresis assay in its alkaline version ph is being increasingly used in genotoxicity testing of substances such as industrial chemicals, biocides, agrochemicals, food additives and pharmaceuticals. Komet software allows the capture and analysis of images from the comet assay. Your browser does not currently recognize any of the video formats available. Dna strand breaks in individual cells can be detected. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of dna linked to the nuclear matrix. 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. Use of frozen tissue in the comet assay for the evaluation of. The comet assay, also known as single cell gel electrophoresis scre, helps scientists to determine whether there has been deoxyribonucleic acid dna damage to a. Although the comet assay, a procedure for quantitating dna damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. Electrophoresis is then performed to identify dna damage by the migration of small fragments of broken dna away from the nucleus forming a tail to the dense circular comet head of undamaged dna.
This makes comet assay iv the fastest, easiest and most accurate way to score comets to date comet assay iv is supplied with a firewire video camera delivering 3x the resolution of previous models. Cometassay single cell gel electrophoresis assay 4250050. This apoptosis assay protocol explains comet assay protocol to. The in vivo alkaline single cell gel electrophoresis assay, hereafter the comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. There are only few limitations of the singlecell gel electrophoresis assay with regard to. A modified alkaline comet assay for measuring dna repair. Comet assay as an indirect measure of systemic oxidative stress. Comet assay interest group is a free forum for exchange of information on comet assay worldwidejoining this group you will have free access to the latest developments in this field as well as access to archived discussions at nih listserv. Comet assay kit 3 well slides ab238544 is a fast and sensitive kit for the measurement of cellular 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 the.
Use of frozen tissue in the comet assay for the evaluation. One of the major advantages of the alkaline comet assay is the possibility of analyzing dna damage and repair in individual cells. It is typically carried out with and without exogenous metabolic activation, usually by supplementing with induced rat liver s9 fraction as indicated in other assays detailed earlier in this chapter. Special interest group toxicology 4 biological background april172018 statistical analysis of the comet assay. In a comparison of different screening methods, uhl et al. Use of the alkaline comet assay to assess dna repair capacity in human populations has been limited by several factors, including lack of methodology for use of unstimulated cryopreserved peripheral blood mononuclear cells pbmcs, insufficient control of interexperimental variability, and limited analysis of dna repair kinetics. The comet assay is commonly employed in the initial screening of novel chemicals or pharmaceuticals for dnadamaging effects. To determine whether the power of the comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in. Comet assay, or single cell gel electrophoresis assay, is a powerful technique for quantitating dna damage and repair in eukaryotic cells and some prokaryotic cells. The comet assay method, while one of many ways of measuring oxidative stress 18,19, is relatively simple, versatile and inexpensive.
The assay can be used in both invitro and invivo testing and has been shown to be a powerful and sensitive predictor of. Dec 19, 2017 9 videos play all comet assay films simon collins bill wiese man who went to hell 23 minutes in hell condensed duration. Since then, the neutral comet assay is mostly used to detect doublestranded. Is comet assay applicable to stored whole blood samples. Jan 01, 2003 the in vivo alkaline single cell gel electrophoresis assay, hereafter the comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. During the last 30 years, the comet assay has become widely used for the measurement of dna damage and repair in cells and tissues.
The comet assay is a versatile and sensitive method for measuring single and doublestrand breaks in dna. Invitrogen and 20000 cells were used for single cell gel electrophoresis with ikzus comet assay kit 0905050k, ikzus. Rating is available when the video has been rented. The alkaline comet assay is capable of detecting dna double. Click here to visit our frequently asked questions about html5.
The comet assay is a wellpublished method for measuring cellular dna damage. It can usually be performed on materials found in most laboratories. Originally developed by perceptive instruments and now part of the instem solution portfolio, comet assay iv is a live video imaging system designed for those seeking fast, accurate and reproducible comet slide scoring. Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit. Use of the comet assay technique for quick and reliable. The physical, chemical, and toxicological properties of the products contained within the cometassay kit may not have been fully investigated. The in vivo comet assay is often used as a 2 nd in vivo test to followup positive in vitro test results substitution of the udstest. The comet assay helps to measure the single double strand dna breaks, alkali labile sites apurinic apyrimidinic sites, dna crosslinks. Comet assay single cell gel electrophoresis sigmaaldrich. The comet assay performed under alkaline conditions ph is considered the optimal version for identifying agents with genotoxic activity. In the simplest form, cells are embedded in agarose on a microscope slide, immersed in a lysis solution, and then exposed to an electric field to attract.
Comet assay was first developed by swedish scientists ostling and johanson 1. Based on our experience and that of others we recommend the use of the following for reliable and reproducible comet assay results. There is a special version of the neutral assay, with prolonged protease digestion at high temperature, which does apparently only detect double strand breaks. Comet assay protocol natobioradproject natobioradproject.
Comet assay procedure appropriate tissues are collected and processed to a singlecell suspension layered over a slide in a thin film of agarose. The extent of dna damage was evaluated by the comet assay, which was performed under alkaline conditions according to the procedure of singh et al. The comet assay is increasingly used as a means to evaluate dna damage in cultured cells and tissues exposed to chemicals or other environmental stressors 1. The comet assay, which can detect singlestrand breaks ssbs as initial damage and those developed from alkalilabile sites under alkaline condition ph 12. The comet assay is a gel electrophoresis method used to visualize and measure dna strand breaks in individual cells, using microscopy. 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. It has since increased in popularity as a standard technique for evaluation of dna damagerepair. Comet assay is rapid results are available in days, simple to perform, requires small amounts of the test substance 2550 mg, and can be performed in almost any eukaryotic cell different animal target organs. The smaller the dna fragments, the faster their migration toward the anode to form a typical comet with a head composed of intact, undamaged dna and a tail. Comet assay is named due to the formation of comet like tail in the agarose gel after the electrophoresis. Comet assay slide kits the easiest way to get started with the comet assay is to purchase a comet assay kit such as those available from a company like trevigen. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.
During electrophoresis, smaller, damaged dna fragments travel faster toward a positively charged anode 2. Jun 05, 2019 the comet assay, also known as single cell gel electrophoresis scre, helps scientists to determine whether there has been deoxyribonucleic acid dna damage to a single cell from apoptosis cell. Video articles in jove about comet assay include quantification of three dna lesions by mass spectrometry and assessment of their levels in tissues of mice exposed to ambient fine particulate matter, imaging approaches to assessments of toxicological oxidative stress using geneticallyencoded fluorogenic sensors, murine precisioncut liver slices as an ex vivo model of liver biology. Evaluating in vitro dna damage using comet assay protocol jove. Biomedical and biological sciences 56,081 views 11. 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. Aug 06, 2015 the alkaline comet assay is capable of detecting dna doublestrand breaks, singlestrand breaks, alkalilabile sites, dnadnadnaprotein crosslinking, and incomplete excision repair sites. In comet assays, cells are immobilized in a bed of low melting point agarose, on a trevigen cometslide. It is a convenient way to screen for general dna damage, regardless of the source or nature of the damage. 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. The comet assay singlecell gel electrophoresis is a simple method for measuring deoxyribonucleic acid dna strand breaks in eukaryotic cells.
Ap biology 2017 frq 6 student samples college board. Single cell gel electrophoresis scge or the comet assay is a versatile, sensitive yet simple and economical technique. A complete description on single cell gel electrophoresiscomet assay, history, what it measures, equipmentrequirments, principle. We present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. When mastered, this method can be used in all areas of medicine in which oxidative stress plays role 810,20,21. Does the neutral comet assay only detect double strand breaks. Evaluating in vitro dna damage using comet assay protocol. An electric current is applied to the gel that causes dna to move electrophoresis, and. Sources of variability in the comet assay include variations in the protocol used to process the cells, the microscope imaging system and the software used in.
The alkaline comet assay is a sensitive and relatively inexpensive technique for the detection of dna damage and dna repair. Comet assay measurement software system score comets. The comet assay serves as an important tool in early drug development as a mechanistic and genotoxic predictor tice et al. The inclusion of digestion of lesionspecific dna repair enzymes in the procedure allows the detection of various dna base alterations, such as oxidative. In these cases, carcinogenic andor precarcinogenic lesions were seen in the 2year carcinogenicity assays in rodents in. A full explanation about bradford assay, coomassie brilliant blue and the calibration curve duration. Many methods have been used to identify genotoxic substances, including the detection of dna damage, chromosome aberrations, and gene mutations both in vitro and in vivo.