Skip to main content
SpringerLink
Log in
Book cover

Apoptosis and Cancer pp 43–56Cite as

Detection of End-Stage Apoptosis by ApopTag® TUNEL Technique

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1219))

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.

This is a preview of subscription content, log in via an institution.

eBook
USD   39.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   54.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   54.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  2. 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

    Google Scholar 

  3. 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

    Article  PubMed  CAS  Google Scholar 

  4. 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

    Google Scholar 

  5. Majno G, Joris I (1995) Apoptosis, oncosis and necrosis, an overview of cell death. Am J Pathol 146:3–15

    PubMed  CAS  PubMed Central  Google Scholar 

  6. 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

    Article  PubMed  CAS  Google Scholar 

  7. Matassov D, Kagan T, Leblanc J, Sikorska M, Zakeri Z (2004) Measurement of apoptosis by DNA fragmentation. Methods Mol Biol 282:1–17

    PubMed  CAS  Google Scholar 

  8. 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

    PubMed  CAS  Google Scholar 

  9. Walker PR, Kokileva L, LeBlanc J, Sikorska M (1993) Detection of the initial stages of DNA fragmentation in apoptosis. Biotechniques 15:1032–1047

    PubMed  CAS  Google Scholar 

  10. 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

    Article  PubMed  CAS  Google Scholar 

  11. Arends MJ, Morris RG, Wyllie AH (1990) Apoptosis: the role of the endonuclease. Am J Pathol 136:593–608

    PubMed  CAS  PubMed Central  Google Scholar 

  12. Bursch W, Paffe S, Putz B, Barthel G, Schulte-Hermann R (1990) Biochemistry of cell death by apoptosis. Biochem Cell Biol 68:1071–1074

    Article  PubMed  CAS  Google Scholar 

  13. 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

    Article  PubMed  CAS  Google Scholar 

  14. 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

    Article  PubMed  CAS  Google Scholar 

  15. 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

    PubMed  CAS  Google Scholar 

  16. 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

    Article  PubMed  CAS  Google Scholar 

  17. 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

    PubMed  CAS  Google Scholar 

  18. 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

    Google Scholar 

  19. 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

    Article  PubMed  CAS  Google Scholar 

  20. 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

    PubMed  CAS  Google Scholar 

  21. 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

    Article  PubMed  CAS  Google Scholar 

  22. 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

    Article  PubMed  CAS  Google Scholar 

  23. 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

    Article  PubMed  CAS  Google Scholar 

  24. 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

    Article  PubMed  CAS  Google Scholar 

  25. 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

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. EMD Millipore, 28820 Single Oak Drive, Temecula, CA, 92590, USA

    Chandra Mohan, Kevin Long, Manpreet Mutneja & Jun Ma

Authors
  1. Chandra Mohan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kevin Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manpreet Mutneja
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jun Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chandra Mohan .

Editor information

Editors and Affiliations

  1. Department of Obstetrics, Gynecology, and Reproductive Sciences,, Yale University, New Haven, Connecticut, USA

    Gil Mor

  2. Department of Obstetrics, Gynecology, and Reproductive Sciences,, Yale University, New Haven, Connecticut, USA

    Ayesha B. Alvero

Rights and permissions

Reprints 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

Publish with us

Policies and ethics

Search

Navigation