Abstract
In 1992 Gavrieli et al. published a seminal article showing that apoptotic cells could be detected by an in situ assay (1). The labeling method depends on the ability of terminal deoxyribonucleotidyl transferase to add nucleotides to breaks in DNA, and has generally been termed TUNEL (terminal dUTP nick end labeling), although ISEL (in situ end labeling) would be a more appropriate description. During the late stages of apoptosis, nucleases are activated that cleave DNA with the production of double-strand breaks. Terminal transferase is used in this in situ assay for apoptosis to add labeled nucleotides to the 3′ ends of DNA molecules, thus providing a sensitive assay for detecting apoptotic cells in tissues. Since the publication of this method, this work has been cited in over 4,300 publications, attesting to the usefulness of the assay.
Similar content being viewed by others
References
Gavrieli Y., Sherman Y., and Ben-Sasson S. A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. CellBiol. 119, 493–501.
Charriaut-Marlangue C., and Ben-Ari Y. (1995) A cautionary note on the use of the TUNEL stain to determine apoptosis. Neuroreport 7, 61-4.
Negoescu A., Lorimier P., Labat-Moleur F., Drouet C., Robert C., Guillermet C., Brambilla C., and Brambilla E. (1996) In situ apoptotic cell labeling by the TUNEL method: Improvement and evaluation on cell preparations. J. Histochem. Cytochem. 44, 959-68.
Steigerwald S. D., Pfeifer G. P., and Riggs A. D. (1990) Ligation-mediated PCR improves the sensitivity of methylation analysis by restriction enzymes and detection of specific DNA strand breaks. Nucleic Acids Res. 18, 1435–1439.
Staley K., Blaschke A. J., and Chun J. (1997) Apoptotic DNA fragmentation is detected by a semiquantitative ligation-mediated PCR of blunt DNA ends. Cell Death Differ. 4, 66–75.
Walker P. R., and Sikorska M. (1994) Endonuclease activities, chromatin structure, and DNA degradation in apoptosis. Biochem. Cell Biol. 72, 615–623.
Sollner-Webb B., Melchior W., Jr., and Felsenfeld G. (1978) DNAase I, DNAase II and staphylococcal nuclease cut at different, yet symmetrically located, sites in the nucleosome core. Cell 14, 611-27.
Lutter L. C. (1979) Precise location of DNase I cutting sites in the nucleosome core determined by high resolution gel electrophoresis. Nucleic Acids Res. 6, 41–56.
Cusick M. E., Wassarman P. M., and DePamphilis M. L. (1989) Application of nucleases to visualizing chromatin organization at replication forks. Meth. Enzymol. 170, 290–316.
Peitsch M. C., Muller C., and Tschopp J. (1993) DNA fragmentation during apoptosis is caused by frequent single-strand cuts. Nucleic Acids Res. 21, 4206-9.
Wolffe A. P. (1995) Chromatin: Structure and Function. Academic Press, New York.
Holtzman E. (1989) Lysosomes. Plenum Press, New York.
Van Dyck J. M., and Wattiaux R. (1968) Distribution intracellulaire de l’exonuclease acide dans le foie de rat. Eur. J. Biochem. 7, 13–20.
Majno G., and Joris I. (1995) Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol. 146, 3–15.
Campagne M. V., Lucassen P. J., Vermeulen J. P., and Balazs R. (1995) NMDA and kainate induce internucleosomal DNA cleavage associated with both apoptotic and necrotic cell death in the neonatal rat brain. Eur. J. Neurosci. 7, 1627–1640.
Rink A., Fung K. M., Trojanowski J. Q., Lee V. M. Y., Neugebauer E., and Mcintosh T. K. (1995) Evidence of apoptotic cell death after experimental traumatic brain injury in the rat. Am. J. Pathol. 147, 1575–1583.
Hu G. (1993) DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3′ end of a DNA fragment. DNA Cell Biol. 12, 763–770.
Marchuk D., Drumm M., Saulino A., and Collins F. S. (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19, 1154–1156.
Didenko V. V., and Hornsby P. J. (1996) Presence of double-strand breaks with single-base 3′ overhangs in cells undergoing apoptosis but not necrosis. J. Cell Biol. 135, 1369–1376.
Wyllie A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-6.
Didenko V. V., Tunstead J. R., and Hornsby P. J. (1998) Biotin-labeled hairpin oligonucleotides. Probes to detect double-strand breaks in DNA in apoptotic cells. Am. J. Pathol. 152, 897–902.
Didenko V. V. Boudreaux D. J. and Baskin D. S. (1999) Substantial background reduction in ligase-based apoptosis detection using newly designed hairpin oligonucleotide probes. BioTechniques 27 1130–1132
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Hornsby, P.J., Didenko, V.V. (2002). In Situ DNA Ligation as a Method for Labeling Apoptotic Cells in Tissue Sections. In: Didenko, V.V. (eds) In Situ Detection of DNA Damage. Methods in Molecular Biology, vol 203. Humana Press. https://doi.org/10.1385/1-59259-179-5:133
Download citation
DOI: https://doi.org/10.1385/1-59259-179-5:133
Publisher Name: Humana Press
Print ISBN: 978-0-89603-952-0
Online ISBN: 978-1-59259-179-4
eBook Packages: Springer Protocols