The power, ease and speed of analysis can be especially appreciated. Opencomet helps to overcome the current limitations in image. Our comet assays provide a fast, convenient way to screen for general dna damage, regardless of the source. The comet assay is a dna damage quantification method that is efficient and easy to perform, and has low timebudget demands and high reproducibility. The assay is wellestablished for measuring dna damage levels in mammalian cells. Our tacs trevigen apoptotic cell systems products have been widely used. The software applies state of the image processing methods to automatically locate and score comets to characterize and quantify the degree of dna damage revealed by the comet assay. Dna fragmentation was measured by the alkaline comet assay, which is known to detect both single and double strand breaks in a wide variety of cell types 6. Trevigen is recognized internationally for the commercialization and standardization of the comet assay. Comet assay for cnidarian cells national oceanic and. Trevigen comet analysis software is a platform that is capable of evaluating a large number of cells as well as generating its corresponding summary statistics. What is the best software for analysis of comet assay stained. 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. Trevigen comet analysis software training video youtube.
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. Undamaged cells had the appearance of the comet tail moment tm analyzer as comet assay parameter and image should analyze using comet image analyzer and data capture software. Lysis solution suitable for either alkaline or neutral comet assay. 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. The slide saves time by eliminating the preparation steps usually associated with the. The standard alkaline comet assay procedure was performed using alkaline cometassay control cells cc, treated with increasing, proprietary concentrations of etoposide that were embedded in low melting agarose and immobilized on 2, 3 and. Jan 09, 2014 the comet assay, in specific, is widely used for measuring oxidative and other types of dna damage. Opencomet is a free software tool for measuring dna damage in comet assay images. To evaluate the degree of damage, the control cells were processed using the. The use of cometslides shortens assay time, and allows the. Our cometassay product line is the only complete and optimized commercially available platform for the comet assay, which include reagents, kits, equipment and analysis software. Cometslides allow direct application of comet lmagarose without base layers or pretreatment. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparibtemozolomide combination therapy to u251 glioma cells. Hydrogen peroxide o neutral comet 40 30 20 10 to tl t2 h202 treatment bleomycin treatment t3 treatment 1 800 trevigen bleomycin healthy 20 e 15 bleomycin hydrogen peroxide neutral comet hydrogen peroxide healthy increase in damage only seen with increasing concentration of bleomycin figure 9 trevigen, inc.
New trevigen software revolutionizes comet assay analysis. Trevigen has a wide range of products for measuring oxidative stress and dna damage. 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 analysis software this program is the fastest way to accurately assess comets, designed by trevigen. Opencomet is an opensource software tool providing automated analysis of comet assay images. Standard alkaline comet assay slides were prepared as follows or obtained directly. 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. Molecular cytogenetics herbert irving comprehensive. The results obtained with our tool were compared with that of fully automatic shareware. Nowadays, it is the most used assay in in vitro assessments and is second. Current ready to use assay including for both human and mouse cells phosphorylated h2ax. To determine relative double stranded dna damage in treatments of a single cell type of cnidarians cells using the comet assay. Data can be exported and analyzed using statistic tools. These cryopreserved cell preparations contain different levels of dna damage to be used as controls when performing the comet assay.
Comet assay single cell gel electrophoresis sigmaaldrich. Casplab comet assay software project lab is a crossplatform image analysis software to measure level of dna damage in. Background trevigens cometassay, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating dna damage in cells. The standard alkaline comet assay was performed using alkaline cometassay control cells cc, treated with. Trevigen s comet analysis software can rapidly evaluate large numbers of cells, related. The comet assay or single cell gel electrophoresis assay is based on the alkaline lysis of labile dna at sites of damage. Trevigen will submit a phase ii sbir application to continue the development of this technology. The results obtained with our tool were compared with that of fully automatic shareware software. The comet assay, in specific, is widely used for measuring oxidative and other types of dna damage. For over 10 years the trevigen research and development team has been improving the technology, creating optimized instrumentation, reagents, and assay protocols. Prior to comet analysis, the identity of trapped cells is confirmed by antiintegrin beta1 immunostaining labeled with quantum dots. The resulting image that is obtained resembles a comet with a distinct head and tail.
Representative results in this first example, we describe the approach for quantifying initial levels of dna damage in human. Automated imaging and analysis of a novel comet assay to enable. Unlike other electrophoresis units on the market, the trevigen cometassay es was designed specifically and only for the comet assay. The comet assay, also known as singlecell gel electrophoresis scge, has become an attractive assay in the assessment of dna damage. The comet assay helps to measure the single double strand dna breaks, alkali labile sites apurinic apyrimidinic sites, dna crosslinks.
It includes various functionalities allowing to determine tail length, area and moment, fragmentation and integrated cell intensity. The comet assay was first introduced by ostling and johanson in 1984. The comet analysis software can rapidly evaluate large numbers of cells and generate summary statistics, increasing processivity approximately 10,000% over. A strong, vibrant research and development group is the lifeblood of all industries. Casplab comet assay software project lab is a crossplatform image analysis software to measure level of dna damage in scge single cell gel casplab comet assay software project browse casp1. Bleomycin treatment t3 1 800 trevigen bleomycin healthy e 30 10 bleomycin alkaline comet hydrogen peroxide healthy increase in damage. Sources of variability in the comet assay include variations in the protocol. Demonstrate advantages of trevigen comet analysis software. The comet assay is a gel electrophoresis method used to visualize and measure dna strand breaks in.
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. Only basal keratinocytes are included in comet data analysis. In this study, we present a semiautomatic comet assay analysis tool that was developed in our laboratory. Casplab comet assay software project lab is a crossplatform image analysis software to measure level of dna damage in scge single cell gel electrophoresis method called also comet assay method. The head is composed of intact dna, while the tail consists of singlestrand or doublestrand dna breaks. I had no difficulty running the imagej program and got the result like the first picture attached. Its simplicity, sensitivity, speed and economy make it a prime. The unwound, relaxed dna is able to migrate out of the cell during electrophoresis and can be visualized by sybr gold i nucleic acid gel stain. Hydrophobic barrier allows easy application of cells directly to slide, and treatment with any of several trevigen dna repair enzymes.
Hydrophobic barrier allows easy application of cells directly to slide, and treatment with any of several. 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. The extended use of the comet assay and the increasing need for genotoxicity testing of chemicals led to the development of the in vivo mammalian alkaline comet assay protocol, now recommended by the international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use and the european food safety. We validated opencomet on neutral comet assay images, which present different characteristics from alkaline comet assay images.
Whether using the cometslide or the cometchip, we recommend the use of trevigens. Download casplab comet assay software project for free. The comet assay, also known as singlecell gel electrophoresis scge, is a simple, sensitive and reliable method for studying dna damage induced by physical and chemical agents. 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 comet analysis software this program is the fastest.
A list of commercially available software package is available from trevigen. Comet assay does not require any special apparatus or chemicals. Horizontal electrophoresis chamber, trevigen, cometassay es ii. The software applies state of the image processing methods to automatically locate and score comets to characterize and quantify the degree of dna damage. Trevigen develops technology to advance genotoxicity skin. Comprehensive measurements and results easily graph and report your data. 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. The application is compatible with 4x and 10x images. Standard alkaline comet assay slides were prepared as follows or obtained directly from trevigen. The cometassay product line is the only complete and optimized commercially available platform for the comet assay, which include reagents, kits, equipment and analysis software. This eliminates the time consuming and unreliable traditional method of preparing base layers of agarose. Microsoft layer for unicode on windows 95, 98, and me systems, 1. It can usually be performed on materials found in most laboratories.
The availability of powerful automated and free comet assay imaging software is currently one of the major limitations in using the assay. Select the appropriate set of control cells when performing either the alkaline or neutral comet assay. The standard alkaline comet assay procedure was performed using alkaline cometassay control cells. Representative results in this first example, we describe the approach for quantifying initial levels of dna damage in human lymphoblastoid cells after exposure to oxidative damage using h 2 o 2. The basic principle of the comet assay method is simple. 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. This was a neutral version of the comet assay, and interestingly, they used quite sophisticated techniques of. Combining a novel comet assay procedure with automated. Apr 24, 2014 april 24, 2014 gaithersburg, md trevigen inc. Nov 21, 20 download casplab comet assay software project for free.
The comet assay is a wellpublished method for measuring cellular dna damage. It is expensive but give good results in respect to quantitation. A cross platform public domain pc image analysis program for the comet assay. The comet assay singlecell gel electrophoresis is a simple method for measuring deoxyribonucleic acid dna strand breaks in eukaryotic cells. Analyze comet images by standard comet assay software such as komet 5. The preliminary results suggest that the presented semiautomatic software is an accurate and reliable tool for evaluation of comet assay. Dna fragmentation associated with oxidative dna damage was visualized using. The principle of the assay is based upon the ability of denatured, cleaved dna fragments to. Cometassay neutral control cells trevigen, usa were used to perform. Additional new antibodies will be tested in the future.
The single cell gel electrophoresis assay scge, also known as comet assay is an uncomplicated and sensitive technique for the detection of dna damage at the level of the individual eukaryotic cell. Automated imaging and analysis of a novel comet assay to. Comet assay iv scoring software in order to quickly and accurately score cells using live video imaging, you will need the comet assay iv scoring software and camera. Which fluorescent dna stain is the best for comet assay. Comet assay results, measurements and data processing. Cometassay control cells for single cell gel electrophoresis. Based on our experience and that of others we recommend the use of the following for reliable and reproducible comet assay results. Choose from the standard 3well comet assay kit or the higherthroughput 96well comet assay kit. The increasing trend in damage is accurately measured by opencomet. The comet analysis software can rapidly evaluate large numbers of cells and generate summary statistics, increasing processivity approximately 10,000% over traditional comet analysis.
The control untreated cells should be used to determine the characteristics of data for a. The comet assay, also known as singlecell gel electrophoresis scge, is a simple. The methods used are mostly based on a commercially available comet assay kit from trevigen inc. Opencomet helps to overcome the current limitations in image analysis, making comet assay a more viable method to detect dna breaks produced by rosrns and other damaging agents. The comet assay was first developed by ostling and johanson in 1984 by.
What is the best software for analysis of comet assay. Evaluating in vitro dna damage using comet assay protocol jove. Evaluating in vitro dna damage using comet assay protocol. The comet assay is an efficient method to detect dna damage including single and. Among the icaw 2015 participants, 19% used the comet assay for in vitro genotoxicity testing of nanoparticles. It is compatible with 4x images which allows for rapid screening of up to 300 comets in seconds when analyzing cometchip. Comet assay procedure appropriate tissues are collected and processed to a singlecell suspension layered over a slide in a thin film of agarose. 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. Jun 27, 2006 the comet assay with multiple mouse organs. The openaccess publication on opencomet is available at.
The comet tail can be scored according to dna content intensity. This entry was posted in casplab news on december 11, 20 by support. In fact, the alkaline comet assay is now often the method of choice if low levels of dna damage are to be detected, e. If an average of 208 comets is disposed wells, the cometchip 96 well produced 20,000 comets per chip. Breakthrough software with unmatched performace, designed to improve, standardize, and make comet analysis easier. Equipment and supplies comet assay kit trevigen, or similar. It is specially engineered to complement and operate as the computational component of trevigen s unique cometassay electrophoresis system. Jun 27, 2006 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. The cometassays cometslide is specially treated to promote adherence of low melting point agarose. Applicable for the analysis of either single or doublestrand dna breaks. Comet assay iv combines accurate, repeatable results with powerful data processing and comprehensive presentation. I did comet assay analysis using software open comet v1. Trevigens cometchip platform high throughput dna damage.