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. Opencomet is an opensource software tool providing automated analysis of comet assay images. Article a twotailed comet assay for assessing dna damage in. The single cell gel electrophoresis assay is an uncomplicated and sensitive technique for the. The original comet paper ostling and johanson, 1984. The comet assay has expanded an internationally wellknown method to assay dna damage in various cell types during the last three decades. Lung inflammation and lack of genotoxicity in the comet and micronucleus assays of industrial multiwalled carbon nanotubes.
Comet assay or single cell gel electrophoresis assay detects single or double strand breaks measured at the individual cell level. Once imaging was complete, cellular analysis was performed using gen5 data analysis software. 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 computerized analysis of the images. The alkaline comet assay single cell gel electrophoresis is the most widely used method for measuring dna damage in eukaryotic cells neri et al. The comet assay is considered an appropriate second in vivo. What is the best software for analysis of comet assay. 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. Alkaline and neutral comet assay profiles of sperm dna. Trevigens comet analysis software can rapidly evaluate large numbers of cells, related. You agree not to use the demonstration for collation of any data used as part of a research project or commercial test or study. Feb 24, 2015 this apoptosis assay protocol explains comet assay protocol to determine apoptosis. Automated imaging and analysis of a novel comet assay to enable.
The twotailed comet assay 2tcomet protocol overcomes this limitation. 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. A twotailed comet assay for assessing dna damage in. An electric current is applied to the gel that causes dna to move electrophoresis, and. Comet assay and foci dna damage quantification metasystems. 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. If the orientation of images differs from this, we recommend first rotating them in imagej or another image processing application, and then analyzing then using opencomet. What is the best software for analysis of comet assay stained with silver nitrate. 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. A modification of the original comet assay was developed for the simultaneous evaluation of dna single strand breaks ssbs and double strand breaks dsbs. Simultaneous detection of dna single and double strand breaks. The twodimensional perpendicular tail comet assay 2tcomet combines nondenaturing and denaturant conditions to the same sperm nucleoid. By comparing the values of mean sd of tail length of the comet assay of120, 140and 160 reached to 4. This apoptosis assay protocol explains comet assay protocol to determine apoptosis.
Blois meadow business centre, steeple bumpstead haverhill, suffolk cb9 7bn united kingdom. Genotoxicity study of ethiopian medicinal plant extracts. Alkaline comet assay using cometchip cultured lymphocyte cells in complete media were added to each. The alkaline comet assay is a sensitive technique for measuring singlestrand breaks in dna of individual mammalian cells. Only 10% of the studies conducted as followup of a positive in vitro mammalian cell assay showed positive comet assay results. Positive comet assay data with clear evidence of tissue toxicity cell apoptosis or necrosis should be interpreted carefully. The comet assay is an extremely sensitive dna damage assay.
The head of the comet is composed of intact dna and the tail consists of the dna damage. Factors influencing the sensitivity of the alkaline comet. Nov 21, 20 download casplab comet assay software project for free. The image analysis and processing for quantitative microscopy were performed using the leica qwin software leica qwin.
Accurate automated comet imaging was validated using cometassay alkaline control cells containing known levels of dna damage, processed using the standard alkaline comet assay on 2 and 3well cometslide slides. Download comet assay iv instem offers a free one week trial of the comet assay iv software. There are numerous software for comet image analysis and the output includes a. 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. Ap biology 2017 frq 6 student samples college board.
No positive comet assay results with compounds having no genotoxic properties. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. Evaluating in vitro dna damage using comet assay protocol. Cells were grown to confluence prior to treatment, as indicated. Accommodates 2, 3, 20 and 96 well slides and maintains correct position during. What is the best software for analysis of comet assay stained. Jan 09, 2014 the availability of powerful automated and free comet assay imaging software is currently one of the major limitations in using the assay. Getting started with comet assay iv is as simple as installing the software and connecting the camera. Comet assay iv is the professional choice for those seeking accuracy, reproducibility and glp compliance getting started with comet assay iv is as simple as installing the software and connecting the camera. The 2tcomet assay is a fast, sensitive, and reliable procedure for the quantification and characterization of dna damage in spermatozoa. This trial is provided freeofcharge strictly for noncommercial evaluation purposes only. Cometassay, cometchip, flare assay and cometassay analysis software. 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 57.
Microsoft layer for unicode on windows 95, 98, and me systems, 1. Opencomet is tested to perform well with various comet shapes and sizes including ones typical for neutral as well as alkaline comet assay. 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. To ensure that the dose range tested includes at least two to three noncytotoxic doses, an lmw dna diffusion assay dose range finder using the same dosing schedule as the comet assay and including at least two animals per dose group may be conducted. Trevigens comet analysis software provides the computational power to efficiently analyze digital. Cells are embedded in agarose and lysed, followed by electrophoresis. Combining a novel comet assay procedure with automated imaging and analysis to enable high throughput genotoxicity testing brad larson1, clare whittaker2, sandra woodgate2. 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 comet assay offers sensitive detection of both single and double stranded breaks. Blois meadow business centre, steeple bumpstead haverhill, suffolk cb9 7bn united kingdom tel. The alkaline comet assay is widely used in various research areas, including biomonitoring, routine genotoxicity assessment, and studies of dna repair processes.
After lysis of the cells typically 1 to 2 hours at 4 c the slides are washed in. Leica microsystems, barcelona, spain to compare the cm and tail lengths generated for each chromosome and whole nucleus. Comet assay iv software scoring system for dna damage and repair instem. This is because casplab automaticly writes each measurement result to archivecache file which is creaded in the same folder where casplab exe file is located.
The software accepts static image of comet assay slide in jpeg format, which has already been acquired by the software of the microscope. The comet assay and its applications in the field of. In this study, we present a semiautomatic comet assay analysis tool that was developed in our laboratory. Trevigens comet analysis software can rapidly evaluate large. The two dimensional perpendicular tail comet assay 2tcomet combines nondenaturing and denaturant conditions to the same sperm nucleoid. In the simplest form, cells are embedded in agarose on a microscope slide, immersed in a lysis solution, and then exposed to an electric.
What is the best software for analysis of comet assay stained with. 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. Image j is semiautomated image analysis software written under. This can be done in a few minutes either by yourself or your it department. Comet assay iv is the professional choice for those seeking accuracy, reproducibility and glp compliance. Alkaline and neutral comet assay profiles of sperm dna damage. The basic principle of the comet assay method is simple. Article a twotailed comet assay for assessing dna damage. We present a procedure for the comet assay, a gel electrophoresisbased method that can be used to measure dna damage in individual eukaryotic cells. Factors influencing the sensitivity of the assay to low doses of radiation were. The program shuts down on windows 7 depending on write permissions to folder where exe file is located. The 2tcomet assay is a fast, sensitive, and reliable.
Beafor using casp on windows 98 you need to install two packages from microsoft. The nucleus of an individual cell is placed on a microscope slide coated with an agarose gel. Opencomet is a free software tool for measuring dna damage in comet. Comet assay ivs unique singleclick scoring method and instant live video technology make it the most efficient and easytouse system available for measuring dna damage using single cell gel electrophoresis. 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 aim was to apply dia for classification of cells from a sample processed by 2tcomet. Double stranded sperm dna breaks, measured by comet assay. Analysis of the dna comet tail and nucleoid shape as well as the. A modification of the original comet assay was developed for the simultaneous evaluation of dna single strand breaks ssbs and double strand breaks dsbs in human spermatozoa.
The method uses fluorescent microscopy to examine dna patterns resulting from alkaline agarose gel electrophoresis of individual cells after staining with propidium iodide. Automated comet assay imaging and dualmask analysis to. Lysis solution suitable for either alkaline or neutral comet assay. I have been searching for the best software to acquire more variables from the comet assay. The comet assay is one of the techniques that has been extensively used to assess dna damage in both the somatic cells and the spermatozoa over the past years.
Comet c20 us10 catalina shows off a compact green coma and two tails in this photo taken this morning nov. Combining the in vivo comet and micronucleus assays. 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. It is believed that the comet assay is still growing in use and has high potential to be used in clinical and cancer research. The openaccess publication on opencomet is available at. The brighter and longer the tail, the higher the level of damage. Comet assay iv tm is the latest in a highly successful series of comet scoring systems originally developed by perceptive instruments, now a part of instem. Download casplab comet assay software project for free. The software calculates the parameters including the length of the comet tail, the percentage of the tailed dna, the tail moment tm and the olive tail moment otm.
The software provides the same analysis options as metafer 1, but it is also available as a standalone system. Essentially, anything that can cause dna damage or denaturation except the factors being researched is to be avoided. Leica qwin software was used leica microsystems, wetzlar. A comet assay variant two tailed comet assay applying both alkaline and neutral comet assay in the same slide by turning it 90. Genotoxicity study of ethiopian medicinal plant extracts on. The total projected area of the comet tail, coupling both tail. How do i adjust casp settings so that it can measure comets that are mostly tail. Metafer 1 is the perfect system for automated, highthroughput comet assay analyses. Comet assay software for measuring dna damage and repair. Therefore, recommendations for combining the two assays in vivo have included i the use of separate positive control dose groups for the two separate assays with at least four to six animals in each. 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. The comet assay is a gel electrophoresis method used to visualize and measure dna strand breaks in individual cells, using microscopy. 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 assay, also called single cell gel electrophoresis scge, is a sensitive and rapid technique for quantifying and analyzing dna damage in individual cells.
Right now the program thinks the middle of the tail is the head. Comet assay is named due to the formation of comet like tail in the agarose gel after the electrophoresis. Evaluation of dna damage in vivo by comet assay and. Kits contain comet slides, reagents, and a fluorescent dye to visualize cells under an epifluorescence microscope.
Materials and methods 2tcomet assay the study was carried out using human mature spermatozoa from five healthy donors. A modification of the original comet assay was developed for the simultaneous evaluation of dna ssb and dsb in human spermatozoa. Applicable for the analysis of either single or doublestrand dna breaks. Ostling and johanson 1984 were the first to quantify dna damage in cells using a microgel electrophoresis technique known as singlecell gel electrophoresis or the comet. The two tailed comet assay 2tcomet protocol overcomes this limitation. In vivo alkaline comet assay and enzymemodified alkaline. 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 results obtained with our tool were compared with that of fully automatic shareware software. The comet assay, also known as singlecell gel electrophoresis scge, has become an attractive assay in the assessment of dna damage.
In a neutral comet assay, the dna is kept as double strands so that the assay is used to detect doublestranded breaks. 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. The preliminary results suggest that the presented semiautomatic software is an accurate and reliable tool for evaluation of comet assay. Unlike the other assays described above, the comet assay measures transient genetic damage that is not a fixed change to the dna. It detects strand breaks sbs and alkalilabile sites at frequencies from a few hundred to several thousand breaks per cella biologically useful range, extending from low endogenous damage levels to the extent of damage that can be. Genotoxic effect of ptrolobium stellatum extracts on hepg 2 cells. It is compatible with 4x images which allows for rapid screening of up to 300 comets in seconds when analyzing cometchip. Combining a novel comet assay procedure with automated.