The assay principle the deadend fluorometric tunel system measures the fragmented dna of apoptotic cells by catalytically incorporating fluorescein12dutpa at 3. Combining other methods with tunel, such as microscopic. Prepare a control incubation buffer without rtdt enzyme by combining 98. Trevigen has developed a series of tunelbased assay kits for the in situ detection of apoptosis with colorimetric and fluorometric options. The comet assay is an extremely sensitive dna damage assay. The tacs tdt kits contain a highly purified form of the tdt enzyme for the enzymatic incorporation of biotinylated nucleotides. Manual counting is also very laborintensive and inaccurate.
Protocol overview for use of the deadend fluorometric tunel. 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. Tunel staining protocol for apoptosis detection enzyme. Enzyme by combining 45l of equilibration buffer, 5l of nucleotide mix and. Dna fragmentation represents a characteristic hallmark of apoptosis. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the apobrduihc tunel apoptosis kit. Nonapoptotic cells do not incorporate significant amounts of the brdutp owing to the lack of exposed 3hydroxyl dna ends. Terminal deoxynucleotidyl transferase dutp nick end labeling tunel is a method for. However, only an observation in light microscopy with high magnification permits the detection of chromatin condensation and apoptotic bodies.
Caution the label solution contains cacodylate, toxic by inhalation and swallowed, and cobalt dichlo ride, which may cause ca ncer by inhalation. Oh termini in singlestranded breaks in highmolecularweight nuclear dna fragments. 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. Tunel assay kits for apoptosis detection biocompare.
Deadend fluorometric tunel system technical bulletin, tb235. 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. Apobrdu tunel assay kit c cell fixation is an important step in analyzing apoptotic samples. Detection of apoptosis in paraffin embedded tissues. 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. 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. Jan 27, 2015 sperm dna damage affects the conception rate resulting from human assisted reproduction technology.
A failure of cells to undergo apoptosis is a common feature of many cancers 2 thus investigation. In this method, the degree of integration of dutp deoxyuridine triphosphate. A cautionary note on the use of the tunel stain to determine apoptosis. Apr 10, 2014 as apoptosis assays go, tunel technology isnt exactly new, dating back to the early 1990s. Apoptotic cells can be detected by terminal deoxynucleotidyl transferase tdtmediated dutp nick end labeling tunel.
As it attaches deoxynucleotides to the 3hydroxyl terminus of dna breaks, tdt is pivotal to the tunel staining method. 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. Tunel staining was performed using the apoptag in situ detection kit chemicon. Hcs apoptosis tunel assay thermo fisher scientific us. Apoptosisterminal transferase dutp nick end labeling. Tunel as a test for sperm dna damage in the evaluation of. Essentially, anything that can cause dna damage or denaturation. 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.
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. Detection of dna fragments in situ using the terminal deoxyribonucleotidyl transferase tdtmediated biotin16dutp nickend labelling tunel assay is now commonly used to investigate apoptosis. The tunel assay also gives one more key piece of information which the annexin v assay doesnt. Trypsinized islet cells of indicated groups were subjected to a tunel assay.
Because every research is unique, wimasis tunel assay tool is engineered with the flexibility to adapt to the needs of every researcher. In a tunel assay, an enzyme known as terminal deoxynucleotidyl transferase tdt identifies nicks, or points of. As apoptosis assays go, tunel technology isnt exactly new, dating back to the early 1990s. 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. Here, we describe a protocol in which cells are treated with tunel reagent and counterstained with hoechst 33342. 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. Protocols that combine the tunel assay and immunohis. The mtt enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured dark purple formazan product. 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. Oh dna ends using the terminal deoxynucleotidyl transferase, recombinant, enzyme rtdt. Nucleotides for application in apoptosis tunel assay jena. Tunel fluorescence assay is a wellestablished, fast, and simple nonradioactive technique to detect and quantify neurons undergoing apoptosis. In situ cell death detection kit, pod sigmaaldrich. In situ cell death detection kit, pod y version 15 content version.
Detection of apoptotic cells in tissue samples currently relies on the tunel assay. The method is based on the ability of tdt to label blunt ends of doublestranded dna breaks independent of a template. Yet the assay remains popular, in part because it meshes well with other cytological techniques. Tunel positive cardiomyocytes show morphological features of apoptosis and the typical ladder pattern in dna. 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. 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. In recent years, image cytometry has become a method widely used in clinical. Apoptosis is an important biological process during development, and for maintaining tissue homeostasis. The tunel assay is one of the most used methods for assessment of dna. Tunel staining allows for visualization and quantification of. 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.
Analysis of apoptosis by cytometry using tunel assay. Pdf combination of tunel assay with immunohistochemistry for. 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. Apoptotic cells are often proved using tunel assay based.
Tunel assay is a common method with highspecificity in the detection of apoptosis loo, 2002. 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. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase tdt mediated dutp nick end labeling tunel assay. This assay uses a nuclear stain to identify cells and a tunel label to measure dna strand breaks. Tunel staining allows for visualization and quantification of apoptotic cells. Evaluation of sperm dna damage in bulls by tunel assay as. 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. Detection and analysis of dna damage in mouse skeletal muscle. Mtt assay principle pdf this is a colorimetric assay that measures the reduction of yellow 34. This property can be used to identify apoptotic cells by labeling the dna breaks with fluorescenttagged. 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.
Apoptosis detection using terminal transferase and biotin16. Tunelpositive cardiomyocytes show morphological features of apoptosis and the typical ladder pattern in dna. A modified protocol should be followed for endlabelling of tissue cryosections and cell preparations fixed on slides see pages 1016 for further. Sperm dna damage affects the conception rate resulting from human assisted reproduction technology. Tunel reaction mixture the tunel reaction mixture should be prepared immedi ately before use and should not be stored. The fluorochromebased tunel assay applicable for flow cytometry, combining the detection of. 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.
Cleavage of genomic dna during apoptosis may yield double stranded as well as. Apoptosisterminal transferase dutp nick end labeling tunel. It requires a minimum of a two color flow cytometer with a green fluorescence 515nm and red fluorescence635nm detector. Procedure for the detection of apoptosis in cultured cells. 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. Mix 45 l tunel label with 5 l tunel enzyme prior to use. Pdf oxidative cell damage causes disruption of dna via formation of. The tunel method for detection of apoptosis and dna damage was first reported over 20. Terminal deoxynucleotide transferase tdt is an enzyme that is expressed in certain immune cells. Evaluation of sperm dna damage in bulls by tunel assay as a. The fixation procedure only takes a little while and is a paraformaldehydeethanol fixation. 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.
Detection of dna fragmentation in apoptotic cells by tunel. The tunel assay, used with appropriate tissue processing techniques and. Apoptosis detection using terminal transferase and biotin16dutp tunel enzyme method apoptosis detectiontunel staining service. Essentially, anything that can cause dna damage or denaturation except the factors being researched is to be avoided. The tunel assay is most commonly used to detect cells undergoing apoptosis, which is a form of programmed cell death. 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. 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. Insulin is stained green and tunelpositive nuclei are stained red. The kits are optimized for hcs and provide a choice of three wavelength options to aid in multiplexing with other cellular measurements. About this assay caymans wst1 cell proliferation assay provides a tool for studying induction and inhibition of cell proliferation in any in vitro model. Terminal deoxynucleotidyl transferase dutp nick end labeling tunel is an established method for detecting dna fragments. The deadend colorimetric tunel system provides reagents for terminal.
Nucleotides for application in apoptosis tunel assay. 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. Concurrently, the avidinperoxidase labeling assay applicable for light absorption microscope was described by gavrieli et al. Alternate fixation methods may be necessary to fully exploit some cell systems. 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. Terminal deoxynucleotidyl transferase dutp nick end labeling is a method for detecting dna fragmentation by labeling the terminal end of nucleic acids.
The tunel assay is considered the gold standard for measuring apoptosis because is works on 95% of cells. Unfixed cells may lose smaller dna fragments, leading to lower signals. Manual of histological techniques and their diagnostic. 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. 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. Clickit tunel alexa fluor imaging assay protocol thermo. 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. 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. Rudimentary assay details have been omitted for the sake of brevity.
The deadend fluorometric tunel system measures the fragmented dna of apoptotic cells by catalytically incorporating fluorescein12dutp at 3. 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. 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. 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. Tunel is listed in the worlds largest and most authoritative dictionary database of abbreviations and acronyms. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results.
Furthermore, the clickit tunel assay allows multiplexing with surface and intracellular biomarker detection. In contrast to tunel, which only stains apoptotic cells, hoechst 33342 stains the dna of all cells. Looking for online definition of tunel or what tunel stands for. 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. The presence of these groups is considered an established marker of apoptosis. Tunel as a test for sperm dna damage in the evaluation of male infertility rakesh k. For each image, combine the muscle area mask and the tunel mask. To avoid the loss of fragmented dna and to allow enzyme and nucleotide entrance, cells need to be fixed and. The assay is based on the enzymatic cleavage of the tetrazolium salt wst1 to formazan by cellular mitochondrial dehydrogenases present in viable cells.
Inhibition of growth and induction of apoptosis in. A cautionary note on the use of the tunel stain to. 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. Tunel label is used in combination with tunel enzyme to prepare the tunel reaction mixture.