In situ cell death detection kit, pod y version 15 content version. Pdf terminal deoxynucleotidyl transferase tdt dutp nickend labeling tunel assay has been designed to detect apoptotic cells that undergo. The tunel assay relies on the presence of nicks in the dna which can be identified by tdt, an enzyme that catalyzes the addition of dutps that are labeled with fluorescein. Clickit plus tunel assay for in situ apoptosis detection. Now that you understand the basic principles and concepts behind the tunel assay, lets outline a general protocol for performing this technique in tissue sections. Jan 27, 2015 the tunel assay can detect dna strand breaks that are mainly induced by reactive oxygen species ros and abortive apoptosis 7, 8.
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 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. In situ cell death detection kit, pod sigmaaldrich. Unfixed cells may lose smaller dna fragments, leading to lower signals. 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 assay is commonly called tunel, the acronym of t erminal deoxynucleotidyl transferasemediated du tp n ick e nd l abeling. Hcs apoptosis tunel assay thermo fisher scientific us. Indirect assays based upon quantal responses all or none. 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. Apoptosis detection using terminal transferase and biotin16dutp tunel enzyme method apoptosis detection tunel staining service. Note that this assay summary represents the use of ab206386 with paraffinembedded tissue sections.
After incorporation of the labeling moiety into dna fragments, detection is achieved through a catalyzed click reaction. Delfia cytotoxicity assay on a plate reader bmg labtech. Here, we describe a protocol in which cells are treated with tunel reagent and counterstained with hoechst 33342. Doses of the standard and test preparations are sufficient to produce a specified response, and can be directly measured. Sdf testing view project research profile view project rakesh k sharma cleveland clinic 458 publications 10,148 citations see profile ashok agarwal cleveland. Principles involved in bioassay by different methods. View full assay principle apoptotic cells can be detected by terminal deoxynucleotidyl transferase tdtmediated dutp nick end labeling tunel.
Tunel fluorescence assay is a wellestablished, fast, and simple nonradioactive technique to detect and quantify neurons undergoing apoptosis. Tunel staining allows for visualization and quantification of apoptotic cells. Apobrdu tunel assay kit c cell fixation is an important step in analyzing apoptotic samples. We then go through a general protocol for performing tunel assays on tissue sections and visualizing the. Analysis by fluorescence microscopy or flow cytometry. If your tunel assay does not fit the requirements above, send us a quick note or. Here, we describe the development of cam xenograft model of hepatocellular carcinoma hcc with embryonic survival rates of up to 93% and reliable tumor take leading.
Fortunately, a number of sperm function tests are available to assess sperm dna integrity. The tunel assay is the most widely used method to detect fragmented dna in apoptotic cells in tissue samples. Titertacs colorimetric apoptosis detection kit trevigen. Tunel staining relies on the ability of the enzyme terminal deoxynucleotidyl transferase to incorporate labeled dutp into free 3. The kits are optimized for hcs and provide a choice of three wavelength options to aid in multiplexing with other cellular measurements. Apoptosis is characterized by the activation of endogenous endonucleases, particularly the caspase3 activated dnase cad, with subsequent cleavage of nuclear dna into internucleosomal fragments of roughly 180 base pairs bp and multiples thereof 360, 540 etc. Tunel assay 14 one of the possible causes of infertility in men with normal semen parameters is abnormal sperm dna. 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. The cam assay allows for the study of tumor growth, antitumor therapies, and protumor molecular pathways in a biologically relevant system that is both cost and timeeffective. Clickit tunel alexa fluor imaging assay protocol thermo.
Dehydrogenasebased assays reflect cell conditions with more sensitivity than the other assays because they depend on several elements including dehydrogenase, nadh, nadph, and mitochondrial activity. The presence of these groups is considered an established marker of apoptosis. Tunel assay and cell cycle distribution with pi cycle. Dissolved meoh, chcl3 and etoac extracts in absolute ethanol and water extract in distilled water. Apoptotic dna fragmentation is a key feature of apoptosis, a type of programmed cell death. The label solution contains cacodylate, toxic when inhaled or swallowed and cobalt dichloride, which may cause cancer by inhalation. Terminal deoxynucleotidyl transferase dutp nick end labeling tunel is an established method for detecting dna fragments. An indicator of apoptosis in imagebased assays subject the understanding of the cellular signaling processes leading to programmed\r\ncell death \ apoptosis \ is of the utmost importance in the study of autoimmune\r\ndiseases such as rheumatoid arthritis, parkinson s, and alzheimer s disease. 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. Several assays are available to monitor apoptosis and other forms of cell death. Avoid exposure and obtain special instructions before use. The tunel assay begins with incorporation of modified dutp at the 3oh end of the fragmented dna.
The tunel assay is widely used to detect apoptosis, implying a specificity for. Tunel assay principle dna fragmentation represents a characteristic hallmark of apoptosis. A doublelabeling assay for detecting apoptotic cells, using the terminal deoxynucleotidyl transferase tdtmediated dutp nick endlabeling tunel assay, and antigens of interest, using. Oh termini in singlestranded breaks in highmolecularweight nuclear dna fragments. The deadend fluorometric tunel system is a classic tunel assay designed for the specific detection and quantitation of apoptotic cells within a cell population. 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 occurs during embryonic development, as well as in maintenance of tissue homeostasis, under pathological conditions, and in aging. An overview of techniques the study of dna damage holds a wide interest within both basic and applied fields of research. Detection of apoptosis by the tunel assay springerlink. Comparison of comet assay, electron microscopy, and flow. Tunel test has not been standardized to the same extent as scsa and few laboratories have attempted to use this test in a clinical setting, limiting its use in patients in past. Detection of dna fragmentation in apoptotic cells by tunel. This study, using a jurkat cell model, initially evaluated.
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. Sufficient material is supplied for 50 coverslips based on the protocol below. Nucleotides for application in apoptosis tunel assay jena. The deadend fluorometric tunel system measures the fragmented dna of apoptotic cells by catalytically incorporating fluorescein12dutp at 3. Terminal deoxynucleotidyl transferase dutp nick end labeling is a method for detecting dna fragmentation by labeling the terminal end of nucleic acids.
Tunel staining was initially described as a method for staining cells that have undergone programmed cell death, or apoptosis. The titertacs colorimetric apoptosis detection kit is a tunel based assay taking advantage of trevigens exclusive in situ labeling technology bringing it to the 96 well microplate format for high throughput quantitative detection of apoptosis. Oh dna ends using the terminal deoxynucleotidyl transferase, recombinant, rtdt enzyme. The major steps of the tunel assay include fixing the tissue of interest, permeabilization of the tissue, adding tunel reagents, stopping the tunel reaction, and finally the analysis. Terminal deoxynucleotidyl transferase dutp nick end labeling tunel is a method for. The assay relies on the use of terminal deoxynucleotidyl transferase tdt, an enzyme that catalyzes attachment of deoxynucleotides, tagged with a fluorochrome or another. Alternate fixation methods may be necessary to fully exploit some cell systems. The in ovo chick chorioallantoic membrane cam assay as an. Cleavage of genomic dna during apoptosis may yield double stranded as well as. The techniques available, including in situ tunel terminal deoxyribonucleotide transferasemediated dutpx nick endlabeling staining, dna ladder assay, and flow cytometry, suffer from low sensitivity or from a high falsepositive rate. 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. Neutral red uptake assay for the estimation of cell. When using the solution do not eat, drink or smoke.
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. 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. Analysis of apoptosis by cytometry using tunel assay. In contrast to tunel, which only stains apoptotic cells, hoechst 33342 stains the dna of all cells. Because every research is unique, wimasis tunel assay tool is engineered with the flexibility to adapt to the needs of every researcher. Some of the authors of this publication are also working on these related projects. Tunel staining the tunel assay is most commonly analyzed by light microscopy. Wst8 assay is a colorimetric assay for the determination of viable cell numbers and can be used for cell proliferation assays as well as cytotoxicity assays. For chromogenic tunel staining, we recommend tunel assay kit hrpdab ab206386. Pdf detection of apoptosis by tunel assay researchgate. This process is experimental and the keywords may be updated as the learning algorithm improves. Assay principle the deadend colorimetric tunel system endlabels the fragmented dna of apoptotic cells using a modified tunel method. The in ovo chick chorioallantoic membrane cam assay as. 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.
Principles and type of damage detected the tunel assay relies on a simple principle. Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. Horseradish peroxidaselabeled streptavidin streptavidin hrp is then. This system measures nuclear dna fragmentation, an important biochemical hallmark of apoptosis in many cell types, providing simple, accurate and rapid detection of apoptotic cells.
Dna fragmentation represents a characteristic hallmark of apoptosis. Therefore, the tunel assay may be the most appropriate method for assessing sperm dna strand breaks 8, 11. Since the introduction of terminal deoxynucleotidyl transferasedutp nick end labeling tunel assay in 1992, 1 the tunel assay is the most widely used in situ test for apoptosis study. Tunel staining protocol for apoptosis detection enzyme. Test principle apoptotic cells can be detected by terminal deoxynucleotidyl transferase tdt mediated dutpbiotin nickend labeling tunel. Tunel ap is used for the conversion of fluorescence based tunel detection. How other researchers have used fitc tunel assay kit ab66108. The neutral red uptake assay is one of the most used cytotoxicity tests with many biomedical and environmental applications 1,2,3,4,5,6, as shown in. In addition we discuss the results of this assay in light of the issues risen above. In indirect bio assays the relationship between the dose and response of. This is a colorimetric assay that measures the reduction of yellow 34,5dimethythiazol2yl2,5diphenyl tetrazolium bromide mtt by mitochondrial succinate dehydrogenase. This tunel assay kit has been used in publications in a variety of sample types, including.
The deadend colorimetric tunel system endlabels the fragmented dna of apoptotic cells using a modified. And due to the size of the fluorophore, the modified dutp can display. Detection of apoptosis by tunel assay springerlink. Sperm dna strand breaks occur in each ejaculate and can also be induced by ros 9, 10.
Nucleotides for application in apoptosis tunel assay. Find out more about the tunel method in the tunel staining tunel assay guide. 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. 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. This colorimetric assay uses reduction of a yellow tetrazolium salt 34,5 di methyl thiazol 2yl2,5di phenyl tetrazolium bromide, or mtt to.
The tunel assay is most commonly used to detect cells undergoing apoptosis, which is a form of programmed cell death. These assays focus on changes that occur during apoptosis the annexin v and propidium iodide assays are based on changes in membrane properties and permeability, and the tunel assay. An assay that relies on detection of dna strand breaks dsbs in situ by labeling them with fluorochromes has been developed to identify and quantify apoptotic cells by fluorescence microscopy or cytometry 35. Evaluation of sperm dna damage in bulls by tunel assay as a. Here, we describe an optimized tunel assaybased protocol to assess. 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 viability assay most commonly used throughout the world is the mtt assay, first described by tim mosmann in 1983. Tunel assay science exchange lets you compare quotes from over 10 leading service providers. The formamidemoab technique can be performed by using the protocol of.
The dutp modification is often the addition of a fluorophore. To avoid the loss of fragmented dna and to allow enzyme and nucleotide entrance, cells need to be fixed and. Tunel assay and cell cycle distribution with pi date. The major difference between cck8 and the mtt assay, other than mtts toxicity, is the enzymes involved. Tunel assay terminal deoxynucleotidyl transferase nick translation negative control sample tunel method these keywords were added by machine and not by the authors.
Diluted each sample for at least 5 concentrations twofold dilutions. Tunel as a test for sperm dna damage in the evaluation of. Tunel staining protocol for apoptosis detection enzyme method. The method is based on the ability of tdt to label blunt ends of doublestranded dna breaks independent of a template. Wst8 22methoxy4nitrophenyl34nitrophenyl52,4disulfophenyl2 h tetrazolium, monosodium salt, a highly stable and watersoluble wst, is utilized in cell counting kit8. Detection of apoptosis using the bd annexin v fitc assay. Apoptosis is an important biological process during development, and for maintaining tissue homeostasis. The mtt enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured dark purple formazan product. In situ cell death detection kit, tmr red y version 12 1. As a result, this assay can be combined with the analysis of cell attributes that can require prior cell permeabilization such as dna content measurement, dna fragmentation tunel assay, etc 11, 12, 17.
153 211 3 266 597 514 1107 679 239 838 7 826 252 558 764 1461 1419 58 1325 1029 549 767 1109 949 1325 910 745 260 1356 115 1122