As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. 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. 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 measures dna breaks, induced by different factors. Comet assay results, measurements and data processing. Unlike the other assays described above, the comet assay measures transient genetic damage that is not a fixed change to the dna. The comet assay is a highly sensitive method to identify breaks in the dna of an individual sperm. 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. Single cellsnuclei embedded in agarose on a slide are lysed with detergent and high salt concentration. An electric current is applied to the gel that causes dna to move electrophoresis, and. 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 process offers a way to determine the sensitivity of tumors to radiotherapy or. The physical, chemical, and toxicological properties of the products contained within. Automatic analysis of slides processed in the comet assay. In comet assays, cells are immobilized in a bed of low melting point agarose, on a trevigen cometslide. Combining a novel comet assay procedure with automated. Do not pour solutions directly over slides or drop slides i. Measuring dna repair comet assay is an important determinant of individuals capacity for dna repair and their susceptibility to cancer. The use of trevigens cometslide shortens assay time and allows the rapid and reliable analysis of large numbers of samples. This lysis step digests the cellular and nuclear membranes and allows the release of coiled dna loops generally called nucleoids and dna fragments. Please use one of the following formats to cite this article in your essay, paper or report. 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. After image analysis the damage in dna looks like acomet, so thats why it is known as comet assay.
Validation of the 3d skin comet assay using full thickness. Comprehensive measurements and results easily graph and report your data. The comet assay single cell gel scg electrophoresis or comet assay is a rapid and very sensitive fluorescent microscopybased method to examine dna damage and repair at the level of individual cells. 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 nucleus of an individual cell is placed on a microscope slide coated with an agarose gel. Applications of the comet assay since the initial development of the comet assay, efforts have been made to improve assay sensitivity and reliability, extend applications to the analysis of various types of dna damage in various cell types79, increase samplehandling capacity10,11, and standardize the protocols and analysis12.
Comet assay or single cell gel electrophoresis assay detects single or double strand breaks measured at the individual cell level. 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. If more sperm display damaged dna with the comet assay, the chances of achieving a successful pregnancy, either spontaneous or through in. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. The comet assay for dna damage and repair springerlink. 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 uds test. This test guideline is therefore based on the comet assay method protocol 12, which w a s d e v e l o p e d. Comet assay kit 3well slides ab238544 is a fast and sensitive kit for the measurement of cellular dna damage. The comet assay is a wellpublished method for measuring cellular dna damage. The mechanism of formation of comets under neutral or alkaline conditions is best. Alkaline unwinding solution, ph 200 mm naoh, 1 mm edta wear gloves when preparing and handling the alkaline unwinding solution.
Pdf the comet assay is a sensitive and rapid method for dna strand break detection in individual cells, and the year 2009 represents the 25th. Comet assay as an indirect measure of systemic oxidative. 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. The comet assay serves as an important tool in early drug development as a mechanistic and genotoxic predictor tice et al. 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. We present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. Support protocol 3 describes a procedure to cryopreserve whole blood samples for future comet assays. For the comet assay we used the standard procedure originally described by singh et al. The comet assay is a gel electrophoresis method used to visualize and measure dna strand breaks in individual cells, using microscopy. Comet assay is named due to the formation of comet like. The head is composed of intact dna, while the tail consists of singlestrand or doublestrand dna breaks. The openaccess publication on opencomet is available at. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. 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 represents a widespread technique for simple, sensitive,fast and economic measurement of dna damage on a single cell level both in vitro and in vivo. Comet assay is ideal for investigating nutrient or micronutrient effects at the level of dna damage in humans. Comet assay single cell gel electrophoresis sigmaaldrich. Comet assay iv combines accurate, repeatable results with powerful data processing and comprehensive presentation. We present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual. 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. Pdf the alkaline comet assay single cell gel electrophoresis is the most widely used method for measuring dna damage in eukaryotic cells neri et. Materials in assay methods invitro invivo limititaions 2. Overview comet assay kit 3well slides ab238544 is a fast and sensitive kit for the measurement of cellular dna damage. The comet assay was first introduced by ostling and johanson in 1984.
This solution may be refrigerated at oecd guideline for the testing of chemicals in vivo mammalian alkaline comet assay introduction 1. Introduction combining a novel comet assay procedure with automated imaging and analysis to enable high throughput genotoxicity testing brad larson1, clare whittaker2, sandra woodgate2, and peter banks1 1biotek instruments, inc. Once the comet assay procedure and image preparation were complete, multiple calculations were immediately performed using gen5 microplate reader and imager software, including commonly used assay metrics on an individual comet basis. The comet assay is a method for measuring dna strand breaks in eukaryotic cells. Ap biology 2017 frq 6 student samples college board. Trevigens 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. Although the majority of our customers use one of the following fluorochromes for their comet slides.
This was a neutral version of the comet assay, and interestingly, they used quite sophisticated techniques of image analysis for quantification of the comets, using acridine orange ao as the dna binding dye fig. Single cell gel electrophoresis scge or the comet assay is a versatile, sensitive yet simple and economical technique. 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. Comet assay techniques can also be used in the screening of compounds for use in cancer medication for patients. This apoptosis assay protocol explains comet assay protocol to determine apoptosis. Is the comet assay a sensitive procedure for detecting. Once you have obtained your results, you can easily process them using the spreadsheet generator, which is included free with every comet assay iv system. 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 following 3d skin comet assay protocol reflects the standard operating. The comet assay is a single cell gel electrophoresis assay scge for simple evaluation of cellular dna damage. This unit describes alkaline comet assay procedures for assessing dna strand breaks and oxidative base alterations.
Single cellsnuclei embedded in agarose on a slide are lysed. The homogenized liver tissue centrifuged, the supernatant discardes, resuspende the cells at. The resulting image that is obtained resembles a comet with a distinct head and tail. Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit. Jurkat cells untreated left and treated right with etoposide before performing oxiselect comet assay under alkaline electrophoresis conditions at. 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. International validation of the in vivo alkaline comet assay for the detection of genotoxic carcinogens version 14.
An alkaline singlecell gel electrophoresis comet assay. Automated comet assay imaging and dualmask analysis to. Furthermore, the assay is cheap, has low material requirements and can use virtually any eukaryotic cell. The comet assay singlecell gel electrophoresis is a simple method for measuring deoxyribonucleic acid dna strand breaks in eukaryotic cells. At the same time, other groups of scientists expressed a wish.