Terminal deoxynucleotidyl transferase dutp nick end labeling tunel is an established method for detecting dna fragments. Cell death elisa was employed to quantify the nucleosome production result from nuclear dna fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. Apoptotic cells can be detected by terminal deoxynucleotidyl transferase tdtmediated dutp nick end labeling tunel. Jan 27, 2015 sperm dna damage affects the conception rate resulting from human assisted reproduction technology. The tunel assay is most commonly used to detect cells undergoing apoptosis, which is a form of programmed cell death. Tunel as a test for sperm dna damage in the evaluation of male infertility rakesh k. The fluorochromebased tunel assay applicable for flow cytometry, combining the detection of.
The assay relies on the use of terminal deoxynucleotidyl transferase tdt, an enzyme that catalyzes attachment of deoxynucleotides, tagged with a fluorochrome or another marker, to 3hydroxyl termini of dna double strand breaks. Because every research is unique, wimasis tunel assay tool is engineered with the flexibility to adapt to the needs of every researcher. Tunel reaction mixture the tunel reaction mixture should be prepared immedi ately before use and should not be stored. Manual counting is also very laborintensive and inaccurate. Nucleotides for application in apoptosis tunel assay jena.
Apr 10, 2014 as apoptosis assays go, tunel technology isnt exactly new, dating back to the early 1990s. Shop online for a wide selection of promega deadend colorimetric tunel system a modified tunel assay designed for simple, rapid detection of apoptotic cells in situ at the. Cellular viability xtt assay protocol this assay is based on the conversion of the watersoluble xtt 2,3bis2methoxy4nitro5sulfophenyl2htetrazolium5carboxanilide reagent to an orange formazan product by actively respiring cells. Protocols that combine the tunel assay and immunohis. Tunel terminal deoxynucleotidyl transferase dutp nick end labeling assays are a valuable method for detecting dna fragmentation, which is a hallmark of apoptosis, or programmed cell death. Hcs apoptosis tunel assay thermo fisher scientific us. Evaluation of sperm dna damage in bulls by tunel assay as. For each image, combine the muscle area mask and the tunel mask. A cautionary note on the use of the tunel stain to determine apoptosis.
Enzyme by combining 45l of equilibration buffer, 5l of nucleotide mix and. The method is based on the ability of tdt to label blunt ends of doublestranded dna breaks independent of a template. In situ cell death detection kit, pod y version 15 content version. Nonapoptotic cells do not incorporate significant amounts of the brdutp owing to the lack of exposed 3hydroxyl dna ends. A cautionary note on the use of the tunel stain to. Trevigen has developed a series of tunelbased assay kits for the in situ detection of apoptosis with colorimetric and fluorometric options. Terminal transferase dutp nick end labeling tunel assay is a method used to detect dna degradation in apoptotic cells because one of the hallmarks of late stage apoptosis is the fragmentation of nuclear chromatin which results in a multitude of 3hydroxyl termini of dna ends. Tunel label is used in combination with tunel enzyme to prepare the tunel reaction mixture. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase tdt mediated dutp nick end labeling tunel assay. Here, we describe a protocol in which cells are treated with tunel reagent and counterstained with hoechst 33342. Looking for online definition of tunel or what tunel stands for. Terminal deoxynucleotidyl transferase tdt dutp nickend labeling tunel assay has been designed to detect apoptotic cells that undergo extensive dna degradation during the late stages of apoptosis. Sharma, edmund sabanegh, reda mahfouz, sajal gupta, aparna thiyagarajan, and ashok agarwal objectives to standardize the tunel assay by establishing inter and intraobserver variability, interassay. Strong evidence of apoptosis, following ischaemia and epilepsy, has been recently provided by combining genomic dna gel electrophoresis, light and electron microscopy and in situ dnabreak labelling.
Apoptosis detection using terminal transferase and biotin16dutp tunel enzyme method apoptosis detection tunel staining service. As it attaches deoxynucleotides to the 3hydroxyl terminus of dna breaks, tdt is pivotal to the tunel staining method. The assay relies on the use of terminal deoxynucleotidyl transferase tdt, an enzyme that catalyzes attachment of deoxynucleotides, tagged with a fluorochrome or another marker, to 3hydroxyl termini of dna double. In recent years, image cytometry has become a method widely used in clinical. Detection of apoptosis by tunel assay springerlink. Tunel positive cardiomyocytes show morphological features of apoptosis and the typical ladder pattern in dna. The objective of this study was to adapt the terminal deoxynucleotidyl transferasemediated dutp nick end labeling tunel assay to provide a quality parameter for bull semen based on the detection of sperm dna damage. Dapi blue and differential interference contrast dic images were also merged. The clickit tunel alexa fluor imaging assay has been tested in hela, a549, and cho k1 cells with a variety of reagents that induce apoptosis including staurosporine figure 6. In some cell systems apoptosis may progress without internucleosomal fragmentation of dna such cells will be weakly positive in tunel assay and it will be difficult to identify them. To avoid the loss of fragmented dna and to allow enzyme and nucleotide entrance, cells need to be fixed and.
Procedure for the detection of apoptosis in cultured cells. Detection of apoptotic cells in tissue samples currently relies on the tunel assay. One benefit of using a tunel assay for apoptosis research is the ability to detect surface and intracellular biomarkers on the same sample, says guffey. Essentially, anything that can cause dna damage or denaturation. Detection of apoptosis in paraffin embedded tissues. Sperm dna damage affects the conception rate resulting from human assisted reproduction technology. In fact, this acronym is a misnomer because not only dutp but variety of other deoxynucleotides, indirectly or directly fluorochrometagged, are being used in different variants of the tunel assay. This protocol is used for detection and quantification of apoptosis programmed cell death at single cell level, based on labeling of dna strand breaks tunel technology. It requires a minimum of a two color flow cytometer with a green fluorescence 515nm and red fluorescence635nm detector.
The mtt enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured dark purple formazan product. Pdf combination of tunel assay with immunohistochemistry for. Dna fragmentation represents a characteristic hallmark of apoptosis. Pdf oxidative cell damage causes disruption of dna via formation of. Tunel assay kits for apoptosis detection biocompare. The principle of tunel assay relies on terminal deoxynucleotidyl transferase tdtmediated addition of a modified dutp xdutp to 3oh ends of dna fragments that are generated as a result of apoptosis induction. Mix 45 l tunel label with 5 l tunel enzyme prior to use. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. However, only an observation in light microscopy with high magnification permits the detection of chromatin condensation and apoptotic bodies. Tunel staining allows for visualization and quantification of apoptotic cells. In situ cell death detection kit, pod sigmaaldrich. The deadend colorimetric tunel system provides reagents for terminal.
Detection and analysis of dna damage in mouse skeletal muscle. A failure of cells to undergo apoptosis is a common feature of many cancers 2 thus investigation. The fixation procedure only takes a little while and is a paraformaldehydeethanol fixation. Tunel staining tunel assay protocols that use a nucleotide directly tagged with a fluorescent dye are faster than indirect methods, which use either an antibody or a streptavidinbiotin complex, as they require less staining steps. Apoptotic cells are often proved using tunel assay based. Apoptosis is an important biological process during development, and for maintaining tissue homeostasis. Terminal deoxynucleotidyl transferase dutp nick end labeling is a method for detecting dna fragmentation by labeling the terminal end of nucleic acids.
The tunel method for detection of apoptosis and dna damage was first reported over 20. The assay is commonly called tunel, the acronym of t erminal deoxynucleotidyl transferasemediated du tp n ick e nd l abeling. This assay uses a nuclear stain to identify cells and a tunel label to measure dna strand breaks. In contrast to tunel, which only stains apoptotic cells, hoechst 33342 stains the dna of all cells. Essentially, anything that can cause dna damage or denaturation except the factors being researched is to be avoided. The tunel assay is considered the gold standard for measuring apoptosis because is works on 95% of cells. Oct 16, 2015 terminal deoxynucleotidyl transferase dutp nick end labeling is a method for detecting dna fragmentation by labeling the terminal end of nucleic acids. This is a colorimetric assay that measures the reduction of yellow 34,5dimethythiazol2yl2,5diphenyl tetrazolium bromide mtt by mitochondrial succinate dehydrogenase. The tunel assay is one of the most used methods for assessment of dna. Apoptosisterminal transferase dutp nick end labeling. Dna fragmentation occurs as one of the final stages of programmed cell death and has long been considered a hallmark of apoptosis as well as one of the defining biochemical events of the pathway. This is one of the advantages of the tunel assay because not only does it tell you if your cells are apoptotic, but it also tells you where in the cell cycle the cells are apoptotic.
Manual of histological techniques and their diagnostic. Analysis of apoptosis by cytometry using tunel assay. Trypsinized islet cells of indicated groups were subjected to a tunel assay. Nucleotides for application in apoptosis tunel assay apoptosis is the process of an intracellular death program leading to characteristic biochemical and morphological changes within a cell that consequently result in cell death 1. Alternate fixation methods may be necessary to fully exploit some cell systems. Inhibition of growth and induction of apoptosis in. Tunel as a test for sperm dna damage in the evaluation of. Nucleotides for application in apoptosis tunel assay. Mtt assay principle pdf this is a colorimetric assay that measures the reduction of yellow 34. Evaluation of sperm dna damage in bulls by tunel assay as a. The invitrogen clickit tunel alexa fluor imaging assay kits are fast and efficient and offer precise, quantitative data even with high levels of apoptotic cells. Tunel fluorescence assay is a wellestablished, fast, and simple nonradioactive technique to detect and quantify neurons undergoing apoptosis.
The standard apobrdu kit au1001 is a two color tunel terminal deoxynucleotide transferase dutp nick end labeling assay for labeling dna breaks and total cellular dna to detect apoptotic cells by flow or image cytometry. Rudimentary assay details have been omitted for the sake of brevity. Terminal deoxynucleotidyl transferase dutp nick end labeling tunel is a method for. The presence of these groups is considered an established marker of apoptosis.
Yet the assay remains popular, in part because it meshes well with other cytological techniques. Tunel assay is a common method with highspecificity in the detection of apoptosis loo, 2002. The comet assay is an extremely sensitive dna damage assay. Clickit tunel alexa fluor imaging assay protocol thermo. Detection of apoptotic cells by tunel staining this protocol describes detection of apoptotic cells by tunel terminal transferasemediated dutp nick end labeling staining in paraformaldehydefixed paraffinembedded ffpe or frozen fs tissue sections using the in situ cell death detection kit from roche and fluorescence detection. As apoptosis assays go, tunel technology isnt exactly new, dating back to the early 1990s. Unfixed cells may lose smaller dna fragments, leading to lower signals. The assay is based on the enzymatic cleavage of the tetrazolium salt wst1 to formazan by cellular mitochondrial dehydrogenases present in viable cells. The kits are optimized for hcs and provide a choice of three wavelength options to aid in multiplexing with other cellular measurements. Necrosis is caused by the cells inability to maintain homeostasis and is characterized by loss of plasma membrane integrity, cell swelling and lysis, random degradation of dna, and lack of. To avoid the loss of fragmented dna and to allow enzyme and nucleotide entrance, cells need to be fixed and subsequently permeabilized prior to the labeling reaction. The deadend colorimetric tunel system provides the reagents to perform terminal deoxynucleotidyl transferasemediated dutp nickend labeling tunel of fragmented nuclear dna to assess apoptosis in situ at the singlecell level in tissue sections or cultured cells. About this assay caymans wst1 cell proliferation assay provides a tool for studying induction and inhibition of cell proliferation in any in vitro model.
In a tunel assay, an enzyme known as terminal deoxynucleotidyl transferase tdt identifies nicks, or points of fragmentation. Dna fragmentation can be detected in situ within the nuclei of fixed cells and tissues where the integrity of the dna and the free 3 hydroxyl groups at the sites of cleavage have been preserved by. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the apobrduihc tunel apoptosis kit. In this method, the degree of integration of dutp deoxyuridine triphosphate. The tacs tdt kits contain a highly purified form of the tdt enzyme for the enzymatic incorporation of biotinylated nucleotides. Tunel staining protocol for apoptosis detection enzyme. Apoptosisterminal transferase dutp nick end labeling tunel. The tunel assay, used with appropriate tissue processing techniques and. Terminal deoxynucleotide transferase tdt is an enzyme that is expressed in certain immune cells. Apobrdu tunel assay kit c cell fixation is an important step in analyzing apoptotic samples. Deadend fluorometric tunel system technical bulletin, tb235.
Detection of dna fragmentation in apoptotic cells by tunel. This property can be used to identify apoptotic cells by labeling the dna breaks with fluorescenttagged. Tunel is listed in the worlds largest and most authoritative dictionary database of abbreviations and acronyms. Oh termini in singlestranded breaks in highmolecularweight nuclear dna fragments. Oh dna ends using the terminal deoxynucleotidyl transferase, recombinant, enzyme rtdt, which forms a polymeric tail using the principle of the tunel tdtmediated dutp nickend labeling assay. The fluorochromebased tunel assay applicable for flow cytometry, combining the detection of dna strand breaks with respect to the cell cycle phase position, was originally developed by gorczyca et al. Caution the label solution contains cacodylate, toxic by inhalation and swallowed, and cobalt dichlo ride, which may cause ca ncer by inhalation.
Cleavage of genomic dna during apoptosis may yield double stranded as well as. Oh dna ends using the terminal deoxynucleotidyl transferase, recombinant, enzyme rtdt. Tunel staining allows for visualization and quantification of. Combining other methods with tunel, such as microscopic. The deadend fluorometric tunel system measures the fragmented dna of apoptotic cells by catalytically incorporating fluorescein12dutp at 3. The tunel assay also gives one more key piece of information which the annexin v assay doesnt. Detection of dna fragments in situ using the terminal deoxyribonucleotidyl transferase tdtmediated biotin16dutp nickend labelling tunel assay is now commonly used to investigate apoptosis. Tunelpositive cardiomyocytes show morphological features of apoptosis and the typical ladder pattern in dna. Concurrently, the avidinperoxidase labeling assay applicable for light absorption microscope was described by gavrieli et al. Prepare a control incubation buffer without rtdt enzyme by combining 98. Protocol overview for use of the deadend fluorometric tunel. Apoptosis detection using terminal transferase and biotin16dutp tunel enzyme method apoptosis detectiontunel staining service. Insulin is stained green and tunelpositive nuclei are stained red. Tunel is a method for detecting apoptotic dna fragmentation, widely used to identify and quantify apoptotic cells, or to detect excessive dna breakage in individual cells.
Apoptosis detection using terminal transferase and biotin16. Necrosis is caused by the cells inability to maintain homeostasis and is characterized by loss of plasma membrane integrity, cell swelling and lysis, random degradation of dna, and lack of an energy requirement or macromolecular synthesis. Furthermore, the clickit tunel assay allows multiplexing with surface and intracellular biomarker detection. In a tunel assay, an enzyme known as terminal deoxynucleotidyl transferase tdt identifies nicks, or points of. Tunel staining was performed using the apoptag in situ detection kit chemicon. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. The assay principle the deadend fluorometric tunel system measures the fragmented dna of apoptotic cells by catalytically incorporating fluorescein12dutpa at 3. A modified protocol should be followed for endlabelling of tissue cryosections and cell preparations fixed on slides see pages 1016 for further.