Opencomet helps to overcome the current limitations in image. The tails were evaluated through comet assay iv analysis software perceptive instruments ltd and camera avt smart view for, fire package. It binds to double stranded dna by intercalating between base pairs. To determine relative double stranded dna damage in treatments of a single cell type of cnidarians cells using the comet assay. Readidrop propidium iodide cell viability dye biorad. In recent years the comet assay or single cell gel electrophoresis assay has. Modified annexin vpropidium iodide apoptosis assay for. The software for automatic comet analysis was exclusively developed inhouse. The readidrop propidium iodide cell viability assay has a number of key benefits and these include. Cells emitting blue fluorescence were hoechst positive while. Heterogeneity in dna damage using the comet assay olive.
Detection of apoptosis using the bd annexin v fitc assay on the bd facsverse system bd biosciences august 2011 introduction apoptosis is a normal genetically programmed process that. Propidium iodide pi and hoechst 33342 double staining in cultured c6 cells 72 hours after 400. Propidium iodide pi is a membrane impermeant dye that is generally excluded from viable cells. The alkaline comet assay is a sensitive technique for measuring singlestrand breaks in dna of individual mammalian cells. Finally, airdried slides were stained with propidium iodide 10 gml and the. The propidium iodide dye exclusion assay is performed in a krebsringerhepes buffer krh. The comet assay is not particularly good at distinguishing apoptosis from necrosis 27, 28, although measuring the degree of dna fragmentation can be helpful. Add 1 ml of propidium iodide staining solution to cell pellet and mix well. Factors influencing the sensitivity of the alkaline comet. Add 50 ul of rnase a stock solution final concentration 0. Propidium iodide pi and hoechst 33342 double staining. I did comet assay analysis using software open comet v1.
Comet assay for cnidarian cells national oceanic and. Annexin v is a member of a calcium and phospholipid binding family of proteins with vascular anticoagulant activity. Optical density parameter obtained from the comet analysis software. Read our flow cytometry protocol for analysis of cell viability using propidium iodide to help with your experiment. Dna strand breakage and dna structure influence staining with. Image analysis, based on a variety of commercial or free software. The comet assay, also called the single cell gel electrophoresis scge. Equipment and supplies comet assay kit trevigen, or similar.
Comet assay single cell gel electrophoresis sigmaaldrich. Ethidium bromide, propidium iodide, sybr green or sybr gold. Variability of the comet assay is a serious issue, whether it occurs from. Propidium iodide pi is membrane impermeant and generally excluded from viable cells. Propidium iodide pi is a redfluorescent cell viability dye which is excluded from live cells with intact membranes, but penetrates dead or damaged cells and binds to dna and rna by intercalating. Pipette gently using reverse pipetting to avoid creating bubbles, which could alter the amount of light that travels through the sample while reading the plates giving inaccurate results. Pi binds to dna by intercalating between the bases with little or no sequence preference. This is a very powerful assay, which allows viability to be measured dynamically over long periods of time. Detection of apoptosis using the bd annexin v fitc assay. After fluorescence staining with propidium iodide, cells with increased dna. Propidium iodide pi is a fluorescent dye that binds to dna. Propidium iodide assay for cell viability biology 3451.
Automatic analysis of slides processed in the comet assay. Read our comet assay microscopy guide see menu on right for more information. Ideal for flow cytometry and cell corting pi is in a pbs solution without preservative easy storage. Regardless of the stain used, there are some important factors to take into account. Briefly, human keratinocytes hacats were irradiated with different doses of. This study focuses on the optimization of the concentration of propidium iodide p. An automated image analysis algorithm extracts, segments and scores comet shapes. Cyprotexs apoptosis and necrosis assay utilises annexin v and propidium iodide dual staining to monitor cellular death. The availability of powerful automated and free comet assay imaging software is currently one of the major limitations in using the assay. The comet assay or single cell gel electrophoresis has proven to be a versatile. Direct use of the comet assay to study cell cycle distribution and its.
The method uses fluorescent microscopy to examine dna patterns resulting from. Apoptosis and necrosis occur during cell death in response to cytotoxic conditions. Propidium iodide viability on a plate reader bmg labtech. Comparing different staining procedures, the intercalating dye propidium iodide. Originally developed to measure variation in dna damage and repair capacity within a population of mammalian cells, applications of the comet assay now range from human and sentinel animal. Propidium iodide cell viability flow cytometry protocol. Controlling variation in the comet assay genetics frontiers. Opencomet is a free software tool for measuring dna damage in comet assay images.
Finally, the slides were stained with propidium iodide 20 gml and. Because pi is excluded from viable cells, it often used to selectively stain dead cells in a mixed livedead cell population. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a. I have used propidium iodide pi and it give good result over etbr. Although the majority of our customers use one of the following fluorochromes for their comet slides. There are also some dyes that will enter live cells and quantitatively bind to dna, these include hoechst 33342, draq5 ab108410 and the dyecycle dyes. The tails were evaluated through comet assay iv analysis software.
Finally, the slides were stained with propidium iodide 20 gml and visualized under a leica dmla model motorized epifluorescence microscope controlled with software for automatic scanning and image digitization leica microsystems. Which fluorescent dna stain is the best for comet assay. In vitro comet assay cyprotex have partnered with gentronix, specialists in genotoxicity screening, to offer the in vitro comet assay. Opencomet is a free software tool for measuring dna damage in comet. Cells are embedded in agarose and lysed, followed by electrophoresis. New application of the comet assay pubmed central pmc. The comet assay is a versatile and sensitive method for measuring single and doublestrand breaks in dna. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. 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 basic principle of the comet assay method is simple. 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.
Opencomet is validated to be accurate with respect to manual scoring. Propidium iodide pi is a popular redfluorescent nuclear and chromosome counterstain. However, it can easily penetrate dead or damaged cells and as such is commonly used for identifying cell viability in a. Dna damage estimation was evaluated by the image analysis software comet. Propidium iodide pi is widely used in conjunction with annexin v to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability 1,2. Propidium iodide staining of cells to assess dna c abcam. The work described in this application note is based on the propidium iodide pi assay. Propidium iodide or pi is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Dna damage and its repair in singlecell suspensions prepared from yeast, protozoa. Syber green can be used for comet assay and then cometscore software can be used to. As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. Opencomet is an opensource software tool providing automated analysis of comet assay images. Propidium iodide pi is a cell impermeable nucleic acid intercalating dye. The comet assay is most frequently used to analyze white blood cells or.
The alkaline comet assay and similar singlecell gel. Then, 1 ml of propidium iodide staining solution 50. The comet assay, also called single cell gel electrophoresis scge, is a. 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. It is also known as pp4, pap i, cpbi, cabp33, vaca.
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