Abstract
DNA fragmentation, the end stage of apoptosis, is the measure of ultimate demise of the cell. A convenient method for examining apoptosis via DNA fragmentation is by the Terminal deoxynucleotidyl transferase (Tdt) dUTP Nick-End Labeling (TUNEL) assay where the DNA strand breaks are detected by enzymatically labeling the free 3′-OH termini with modified nucleotides. ApopTag® kits detect single-stranded and double-stranded breaks associated with apoptosis. This technique is also helpful to distinguish between apoptotic and necrotic cell death where the latter is associated with random DNA fragment lengths producing a DNA smear. Apoptotic cells stained positive with ApopTag® kits are easier to detect and their identification is more certain, as compared to the examination of simply histochemically stained tissues. In addition, quantitative results can be obtained using flow cytometry and apoptotic cells can be differentiated from necrotic cells with greater than tenfold sensitivity.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516
Mohan C (2010) Apoptosis: receptor and mitochondrial gateways to cell death. In: Signal transduction—a short overview of its role in health and disease, 2nd edn. EMD, San Diego, CA, pp 67–71
Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F (1997) Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1–20
Kerr JFR, Harmon BV (1991) Definition and incidence of apoptosis: an historical perspective, chapter 1. In: Tomei LD, Cope FO (eds) Apoptosis: molecular basis of cell death. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 5–29
Majno G, Joris I (1995) Apoptosis, oncosis and necrosis, an overview of cell death. Am J Pathol 146:3–15
Migheli AM, Attanasio A, Schiffer D (1995) Ultrastructural detection of DNA strand breaks in apoptotic neural cells by in situ end labeling techniques. J Pathol 176:27–35
Matassov D, Kagan T, Leblanc J, Sikorska M, Zakeri Z (2004) Measurement of apoptosis by DNA fragmentation. Methods Mol Biol 282:1–17
Brown DG, Sun XM, Cohen GM (1993) Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J Biol Chem 268:3037–3039
Walker PR, Kokileva L, LeBlanc J, Sikorska M (1993) Detection of the initial stages of DNA fragmentation in apoptosis. Biotechniques 15:1032–1047
Walker PR, Weaver VM, Lach B, LeBlanc J, Sikorska M (1994) Endonuclease activities associated with high molecular weight and internucleosomal DNA fragmentation in apoptosis. Exp Cell Res 213:100–106
Arends MJ, Morris RG, Wyllie AH (1990) Apoptosis: the role of the endonuclease. Am J Pathol 136:593–608
Bursch W, Paffe S, Putz B, Barthel G, Schulte-Hermann R (1990) Biochemistry of cell death by apoptosis. Biochem Cell Biol 68:1071–1074
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501
Staley K, Blaschke AH, Chun J (1997) Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends. Cell Death Differ 4:66–75
McGahon A, Bissonnette R, Schmitt M, Cotter KM, Green DR, Cotter TG (1994) Bcr-Abl maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood 83:1179–1187
Chapman RS, Chresta CM, Herberg AA, Beere HM, Heer S, Whetton AD, Hickman JA, Dive C (1995) Further characterization of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukemia cells. Cytometry 20:245–256
Gold R (1994) Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab Invest 71:219–225
Gorczyca W, Gong J, Darzynkiewicz Z (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1–7
Schmitz GG, Walter T, Seibl R, Kessler C (1991) Non-radioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal Biochem 192:222–231
Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J, Darzynkiewicz Z (1992) DNA strand breaks occurring during apoptosis: their early in situ detection by terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int J Oncol 1:639–648
Wijsman JH, Jonker RR, Keijzer R, Van De Velde CJH, Cornelisse CJ, Van Dierendonck JH (1993) A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem 41:7–12
Mundle S, Iftikhar A, Shetty V, Dameron S, Wright Quinones V, Marcus B, Loew J, Gregory S, Raza A (1994) Novel in situ double labeling for simultaneous detection of proliferation and apoptosis. J Histochem Cytochem 42:1533–1537
Labat Moleur F, Guillermet C, Lorimier P, Robert C, Lantuejoul S, Brambilla E, Negoescu A (1998) TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement. J Histochem Cytochem 46:327–334
Strater J, Gunthert AR, Bruderlein S, Moller P (1995) Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells. Histochem Cell Biol 103:157–160
Tornusciolo DR, Schmidt RE, Roth KA (1995) Simultaneous detection of TdT mediated dUTP-biotin nick end labelling (TUNEL)—positive cells and multiple immunohistochemical markers in single tissue sections. Biotechniques 19:800–805
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Mohan, C., Long, K., Mutneja, M., Ma, J. (2015). Detection of End-Stage Apoptosis by ApopTag® TUNEL Technique. In: Mor, G., Alvero, A. (eds) Apoptosis and Cancer. Methods in Molecular Biology, vol 1219. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1661-0_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1661-0_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1660-3
Online ISBN: 978-1-4939-1661-0
eBook Packages: Springer Protocols