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Terminal Deoxynucleotidyl Transferase-Mediated Dutp Nick © ECVAM DB-ALM: Method Summary Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) assay - Summary Drug Discovery and Activity Testing The method is suitable for the determination of the apoptogenic potential of putative antineoplastic drugs. Objective & Application TYPE OF TESTING : Screening LEVEL OF ASSESSMENT : Efficacy, Toxic potential PURPOSE OF TESTING : Drug evaluation Apoptosis (programmed cell death) is a complex and dynamic process, orchestrated by numerous internal and external factors, which plays an essential role in development, immunity, homeostasis and aging. Apoptosis is the major mode of cell death induced by antineoplastic agents. DNA fragmentation is a hallmark of the apoptogenic process and it is considered as a reliable indicator of apoptosis. At the experimental level, the method is used to assess whether a test compound kills cells through a necrotic or an apoptotic process. The test is validated and is predictive of morphological and biochemical apoptosis in practically every human and animal cell system. The endonucleolytic cleavage of chromatin, assessed by Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) assay, always precedes morphological apoptosis, thus the assay is useful as a drug activity test, as a screening test and in mechanistic studies. An in-depth reviews on the TUNEL assay and its use as a measure of chemotherapy-induced apoptosis have been published by Loo (2011) and Wieder (2005). Basis of the Method TUNEL assay detects breaks in DNA strands, through an enzymatic reaction where the loose ends of DNA are capped with a labeled deoxynucleotide (dUTP). The intensity of labeling is directly proportional to the number of breaks and the severity of DNA fragmentation. Primary or stabilized cell cultures growing in suspension or adhering to slides and culture flasks are incubated with the apoptogenic compound for different times. After fixing and permeabilization, the cells are incubated with labeled derivatives of dUTP and deoxynucleotidyl transferase (TdT). TdT adds the labeled derivatives of dUTP to the 3'-OH groups in single and double-stranded DNA nicks. The amount of dUTP incorporated is proportional to the number of double or single strand nicks present in genomic DNA. The percentage of cells containing fragmented DNA relative to those with intact genomic DNA is assessed and expressed as the apoptotic index. In respect to other methods, used for the detection of DNA fragmentation, such as agarose gel electrophoresis or comet assay, TUNEL is a quantitative method for the determination of the percentage of the DNA fragmented. It is sensitive, affording the highest level of sensitivity for the detection of apoptosis in case fluorescein labeled dUTP. It is simple, as various commercially available kits have been developed, and it is rapid when associated to flow cytometry. Experimental Description Biological and Endpoint Measurement: To assess the level of DNA fragmentation induced by an external agent: APOPTOTIC DEATH: DNA fragmentation The culture conditions for each of these cell lines are standard as they grow either in suspension in RPMI 1640 (HL-60, NB4, K562) supplemented with 10% fetal calf serum (FCS) or in adherence in DMEM (SKOV-3, OVCAR-3, A2780) containing 10% FCS. The culture conditions are not critical for the assay. In principle, the TUNEL assay can be performed on any kind of cultured cell line that is compatible with the flow cytometric analysis. a. Cells (approximately 1-5 x 106 in 2-10 ml of serum-containing medium) growing in suspension in T25 flat bottom flasks are harvested. b. The cell suspension is centrifuged at 400 x g for 10 min and washed two times, by resuspension and centrifugation, with 2-10 ml of PBS (phosphate buffered saline)/1% BSA (bovine serum albumin) at https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/method-summaries page 1 / 6 © ECVAM DB-ALM: Method Summary 4° C. c. Following the last washing, cells are supended in 100 ml of PBS/1% BSA and transferred in 1.5 ml Eppendorf tubes d. One hundred ml of freshly prepared paraformaldehyde solution (4% in PBS, pH 7.4) are added to the cell suspension and incubated for 30 min at room temperature. e. To remove the fixative, tubes are centrifuged at 300 x g for 10 min and the supernatant is carefully removed by aspiration with a fine needle. f. Cells are washed with PBS/1% BSA and centrifuged at 300 x g in a table microcentrifuge. g. The cell pellet is resuspended in 100 ml of a permeabilization solution consisting of 0.1% Triton X-100 in 0.1% sodium citrate and incubated for 2 min at 4° C. h. Cells are washed two times with 200 ml of PBS. i. The cell pellet is resuspended in 50 ml of TUNEL reaction mixture and incubated for 60 min at 37° C in a CO2 incubator. j. Cells are washed 2 times in 200 ml PBS and finally resuspended in 250-500 ml PBS. k. The samples are analyzed by flow cytometry. Special Materials Although the procedure described above can be carried out using single reagents of the highest purity available on the market, the use of commercially available kits is recommended. Most of the kits contain all the necessary reagents. In the Laboratory of Molecular Biology of the Mario Negri Institute the “In situ cell death detection kit, fluorescein” was routinely used (Catalog # 1684795 from Boheringer Mannheim ITALIA SpA, viale G.B. Stucchi 110, 20052 Monza, Italy). Several other kits are available from various manufactures. Some examples: Clontech Laboratories, Inc., 4030 Fabian way, Palo Alto CA 94303-4607, USA. ApoAlert DNA fragmentation assay kit. Roche Diagnostics GmbH, Nonnenwald 2, 82372 Penzberg, Germany. In Situ Cell Death Detection Kit, Fluorescein. EMD Millipore Corporation, Billerica, MA, USA. TUNEL Apoptosis Detection Kit Medical Biological Laboratories, CO. Ltd. 5-10 Marunouchi-3-chome, Naka-ku, Nagoya-468-8882 Japan. MEBSTAIN, apoptosis kit DIRECT and MEBSTAIN apoptosis kit II. Kamyia Biomedical Co. 910 Industry Drive, Seattle, WA 98188-USA. DNA fragmentation TUNEL kit. TREVIGEN Inc. 8405 Helgerman Court, Gaithersburg, MD. 20877-USA. TUNEL assay kit. Specific details on the use can be found in the user’s brochure provided with each kit. Test Compounds This method can be used to evaluate the anticancer drugs as organic chemicals, peptides, peptidoglycans or proteins. Modifications Several modifications to the original TUNEL assay are possible. They differ in the type of labeled dUTP used (fluorescein, biotin, digoxigenin etc.) and in the readout method (fluorescence or light microscopic analysis followed by manual counting or video-imaging; automated flow cytometry). In addition, protocols for cells growing in adhesion are available ( e.g Tsutsui et al., 1997; Basolo et al., 1997). Fixed and permeabilized cells are incubated with modified dUTP and TdT for a fixed amount of time, which depends on the cell type and the characteristics of the biological sample used. Following washing of the excess reagents, samples are read. Discussion Advantages: The method is extremely sensitive and it is specific for the detection of single strand and double strand nicks in genomic DNA (Loo, 2011). The assay does not require radioactive components. A large number of samples can be analyzed in parallel. The use of flow cytometry gives quantitative results in a large number of cells and, in combination with good positve and negative controls, usually does not require a set up of experimental point replicates. The whole procedure requires less than four hours to complete. https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/method-summaries page 2 / 6 © ECVAM DB-ALM: Method Summary Fixation of samples allows accumulation, storage and transport of samples. Several kits containing all the components necessary for the completion of the TUNEL assay are commercially available. Limitations: Quantitative analysis of the results requires a Fluorescence Activated Cell Sorter, which is not routinely available in standard laboratories. The reagents are relatively expensive and have a relatively short bench life. Status Known Laboratory Use: The assay is used by most leading laboratories in the Apoptosis field. The Bibliography section below includes a collection of references on the use of the method. They were selected mainly from the literature published between 1980 and 1997, when the datasheet was first defined within a feasibility study coordinated by DB-ALM. The original selection was compiled in 1998 by the EURL ECVAM expert collaborators from the Laboratory of Molecular Biology, Department of Biochemistry and Molecular Pharmacology of the Mario Negri Institute, Milano (Italy). Since then the TUNEL assay has become an industry standard in detection of chemotherapy-induced apoptosis (Wieder, 2005). Together with caspase assays, mitochondrial assays, plasma membrane assays, and other nuclear apoptosis assays (DNA fragmentation, DNA condensation) it is very widely used method in apoptosis research. Contact Details Dr. Enrico Garattini Laboratory of Molecular Biology Mario Negri Institute for Pharmacological Research Via Eritrea 62 Milan 20157 Italy email: [email protected] Bibliography Amico, D., Barbui, A.M., Erba, E., Rambaldi, A., Introna, M., and Golay, J. (2003) Differential response of human acute myeloid leukemia cells to gemtuzumab ozogamicin in vitro: role of Chk1 and Chk2 phosphorylation and caspase 3. Blood 101, 4589–4597 Ayala-Torres S. Moller PC. Johnson BH. Thompson EB. (1997) Characteristics of 25-hydroxycholesterol-induced apoptosis in the human leukemic cell line CEM. Experimental Cell Research 235(1), 35-47 Basolo F. Pollina L. Fontanini G. Fiore L. Pacini F. Baldanzi A. (1997) Apoptosis and proliferation in thyroid carcinoma: correlation with bcl-2 and p53 protein expression. British Journal of Cancer 75(4), 537-41 Benassi L. Ottani D. Fantini F. Marconi A. Chiodino C. Giannetti A. Pincelli C. (1997) 1,25-dihydroxyvitamin D3, transforming growth factor beta1, calcium, and ultraviolet B radiation induce apoptosis in cultured human keratinocytes.
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