K191100 ezclick tunel in situ dna fragmentationapoptosis. Labeling protocol for adherent cells, cell smears, cytospin preparations, and tissues. Detection of apoptosis in paraffin embedded tissues. The sciencell tunel apoptosis assay is used for detection of apoptosis programmed cell death in individual cells based on the terminal deoxynucleotidyl. The amount of dna fragmentation was determined by tunel assay using a commercially available kit in situ cell death detection kit, fluorescein, roche, indianapolis, in, usa, whereby the free 3oh ends of dna are labeled with fluorescein conjugated dutp by the enzyme terminal deoxynucleotidyl transferase. 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. In the present study, we have applied the tunel assay to welldefined models of apoptosis and necrosis as well as after postmortem autolysis in rat liver.
Sharma, edmund sabanegh, reda mahfouz, sajal gupta, aparna thiyagarajan, and ashok agarwal objectives to standardize the tunel assay by establishing inter. L00297 is one of genscrips newly introduced products. In situ cell death detection kit, pod sigmaaldrich. Evaluation of sperm dna damage in bulls by tunel assay as a. Tunel as a test for sperm dna damage in the evaluation of male infertility rakesh k.
In situ cell death detection kit, tmr red sigmaaldrich. 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. Apoptotic cells can be detected by terminal deoxynucleotidyl transferase tdtmediated dutp nick end labeling tunel. Tunel stands for terminal deoxynucleotidyl transferase dutp nick end labeling. Standardization of tunel assay for assessment of sperm dna fragmentation sajal gupta, 1rakesh sharma, and ashok agarwal1. Advantages and disadvantages of tunel staining tunel assay methods. The results indicate that all modes of cell death studied 1465.
Biotooltm tunel apogreen detection kit is a modified fitc12dutp tunel assay designed to provide simple, accurate and rapid detection of apoptotic cells. Tunel assay in 1992,1 the tunel assay has become the most widely used in situ test for the study of apoptosis. We then go through a general protocol for performing tunel assays on tissue sections and visualizing the. The kit can detect fragmented dna in the nucleus during apoptosis. 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.
Cell meter tunel apoptosis assay kit green fluorescence. Caution the label solution contains cacodylate, toxic by inhalation and swallowed, and cobalt dichlo ride, which may cause ca ncer by inhalation. Tdt enzyme is a 50% glycerol stock and will not freeze at 20. Cell meter tunel apoptosis assay kit red fluorescence ordering information storage instrument platform product number. Tunel assay kit fitc ab66108 previously called tunel assay kit in situ direct dna fragmentation provides complete components including positive and negative control cells for conveniently detecting dna fragmentation by fluorescence microscopy or flow cytometry. Dna fragmentation represents a characteristic hallmark of apoptosis. In this modified tunel assay kit, biotinylated nucleotide is labeled at the dna 3. Tunel procedure in bovine embryos university of florida.
Hela cells were transduced with celllight mitochondriarfp, bacmam 2. In contrast to tunel, which only stains apoptotic cells, hoechst 33342 stains the dna of all cells. Avoid subjecting tunel reaction buffer to multiple freezethaw cycles. Tunel assay to assess extent of dna fragmentation and. Incubate with tunel working solution for 30 min to 1 hour at 37c. Sperm dna fragmentation analysis using the tunel assay. Cell meter tunel apoptosis assay kit red fluorescence. With increasing importance of apoptosis, many assays have been developed to detect apoptosis.
Clickit plus tunel assay for in situ apoptosis detection. Detecting apoptosis in drosophila tissues and cells. Detection of dna fragmentation in apoptotic cells by tunel. Materials to be supplied by the user and reagent preparation please read the material list below recommended for using the deadend colorimetric tunel system in your application.
Tunel was found to be a most powerful marker for predicting poor prognosis in salivary malignancies. Oh termini in singlestranded breaks in highmolecularweight nuclear dna fragments. The comet assay is a versatile technique for detecting damage and with adjustments to the protocol can be used to quantify the presence of a wide variety of dna altering lesions damage. Analysis of apoptosis by cytometry using tunel assay.
Cleavage of genomic dna during apoptosis may yield. Tunel staining the tunel assay is most commonly analyzed by light microscopy. Concurrently it correlated with the most important clinical characteristics currently used for prognosis predicting histopathological grade, metastasis o spread, extra capsular spread and stage. Deadend fluorometric tunel systemtuneltdtmediated dutp nickend labeling. The apobrdu tunel assay kit provides the materials necessary to label dna strand breaks for the detection of apoptotic cells, as well as fixed samples of positive and negative control cells for assessing assay performance. The tunel assay protocol uses terminal deoxynucleotidyl transferase tdt to. Analysis by fluorescence microscopy or flow cytometry. A 48bp oligonucleotide was incubated with 30 units of tdt and an equimolar mix of the modified nucleotide with three other nucleotides.
Oh dna ends using the terminal deoxynucleotidyl transferase, recombinant, enzyme rtdt. The three modalities of tunel we tested laboratory protocol, apoptag kit, and boehringer mannheim kit yielded no significantly different results, with the exception of proteinase kpretreated tissue sections fixed with either bouin or methacarn, for which the boehringer mannheim kit proved more sensitive than the two others. Here, we describe a protocol in which cells are treated with tunel reagent and counterstained with hoechst 33342. Tunel assay terminal dutp nickend labeling to detect apoptotic cells. Furthermore, the clickit tunel assay allows multiplexing with surface and intracellular biomarker detection. Protocols this user guide includes protocols to perform the clickit plus tunel imaging assay on ffpe tissue samples or adherent cells grown on coverslips figure 4. Tunel staining protocol for apoptosis detection enzyme. Tunel staining protocol for apoptosis detection enzyme method. Read through the protocol before running the assay as modified sample preparation protocols are required for suspension cell lines, fixed cell. 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. The fluorochromebased tunel assay applicable for flow cytometry, combining the detection of dna strand breaks with respect to the cell cyclephase position, was originally developed by gorczyca et al. Tunel as a test for sperm dna damage in the evaluation of.
The deadend colorimetric tunel system provides reagents for terminal deoxynucleotidyl transferase tdt mediated nick end. Finally, several applications of the assay to current research will be covered. The damage usually detected are single strand breaks and double strand breaks. Detection of apoptosis by tunel assay springerlink. Click it plus tunel assay 3 figure 2comparison of tdt incorporation of several modified nucleotides. Concurrently, the avidinperoxidase labeling assay applicable for light absorption microscope was described by gavrieli et al. Death detection kit can be used in many different assay systems. Interand intralaboratory standardization of tunel assay. Its signal can be easily detected at the popular fitc channel.
In this unit, we describe the interlaboratory and intralaboratory standardization of the tunel assay using a benchtop. Evaluation of sperm dna damage in bulls by tunel assay as. 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. The kit provides all the essential components with an optimized assay protocol. Pdf sperm dna fragmentation analysis using the tunel. Mtt proliferation assay protocol university of san diego. Apoptosis detection using terminal transferase and biotin16dutp tunel enzyme method apoptosis detection tunel staining service.
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. Pdf terminal deoxynucleotidyl transferase tdt dutp nickend labeling tunel assay has been designed to detect apoptotic cells that undergo. Validation of the inter and intralaboratory standardization of the tunel protocol several literature reports have highlighted the need for standardization of the tunel assay across laboratories as well as within a. Detect apoptotic cells the following protocol describes the method for measuring apoptosis in the positive and negative control cells that are provided in the apobrdu tunel assay kit. It is suitable for fluorescence microplate reader, fluorescence microscope, or flow cytometer. Clickit tunel alexa fluor imaging assay protocol thermo. Pdf detection of apoptosis by tunel assay researchgate. In situ cell death detection kit, tmr red kit for detection and quantificatio n of apoptosis programmed cell death at single cell level, based on labeling of dna strand breaks tunel technology. 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. The method is based on the ability of tdt to label blunt ends of doublestranded dna breaks independent of a template. Tunel fluorescence assay is a wellestablished, fast, and simple nonradioactive technique to detect and quantify neurons undergoing apoptosis. In situ detection of fragmented dna tunel assay fails to.
The clickit plus tunel assay detects apoptotic cells in tissue and cultured cell samples through the use of a small, highly specific labeling moiety and a bright fluorescent dye. Tunel assay quantification of apoptosis in peripheral. Trypan blue staining is a simple way to evaluate cell membrane. Sperm dna fragmentation analysis using the tunel assay article in methods in molecular biology clifton, n. It measures nuclear dna fragmentation, an important biochemical indicator of apoptosis by utilizing fitc12dutp. Since it was developed in the 1990s the tunel assay has been widely employed, becoming one of the most popular strategies to investigate the origin, the mechanism, and the clinical meaning of. In addition, because tunel staining investigates dna breaks that occur. The tunel apoptosis detection kit for paraffinembedded tissue sections biotin labeled pod cat. The presence of these groups is considered an established marker of apoptosis. The assay is commonly called tunel, the acronym of t erminal deoxynucleotidyl transferasemediated du tp n ick e nd l abeling. Sperm dna damage affects the conception rate resulting from human assisted reproduction technology. Apoptosis and cell proliferation roche molecular biochemicals.
After incorporation of the labeling moiety into dna fragments, detection is achieved through a catalyzed click reaction. This assay was developed with hela adherent and jurkat suspension cells and can be modified for any cell line. 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. These techniques include acridine orange staining, tunel, cleaved caspase staining, caspase activity assays and assays for mitochondrial fission and permeabilization. Tunel staining is a modern alternative to analyzing the formation of dna fragments during apoptosis using agarose gel electrophoresis, as used in apoptotic dna ladder isolation kit ab65627. Tunel assay terminal dutp nickend labeling to detect apoptotic cells reagents. Apoptotic cells are often proved using tunel assay based. Biovisions apobrduihctm kit is a twocolor tunel terminal deoxynucleotide transferase dutp nick end labeling assay for labeling dna breaks to detect apoptotic cells by immunohistochemistry. Kingsleykallesen m, mukhopadhyay ss, wyszomierski sl, schanler s, schutz g, rosen jm. We then go through a general protocol for performing tunel assays on tissue sections and visualizing the results using fluorescence microscopy. The assays are useful for examining apoptosis in normal development and in response to developmental perturbations and external stresses.
A protocol for a fluorescent tunel assay that measures the fragmented dna of apoptotic cells by catalytically incorporating fluorescein12dutp at 3. Terminal deoxynucleotidyl transferase dutp nick end labeling tunel is an established method for detecting dna fragments. After incorporation of the labeling moiety into dna fragments, detection is achieved through a. 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. In situ cell death detection kit, pod y version 15 content version. Internucleosomal dna fragmentation is a hallmark of apoptosis in mammalian cells.
718 1080 226 286 860 306 895 562 965 164 1322 786 1402 1222 519 613 993 840 966 856 1538 295 1098 705 1405 340 1322 1308 1102 1104 26 470 1318 1041 459 995 414