Advertisement

Detection of DNA Fragmentation in Retinal Apoptosis by TUNEL

  • Francesca Doonan
  • Thomas G. Cotter
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 935)

Abstract

Terminal dUTP nick end labeling (TUNEL) is an invaluable technique used in the study of late-stage apoptosis. The technique is based upon detection of fragmented DNA, a well-recognized characteristic of apoptosis, usually with fluorescent markers. Here, we describe the TUNEL technique (1) employing two different detection techniques, fluorescence microscopy and fluorescence-activated cell sorting (2) which can be applied to the analysis of apoptosis in retinal tissues or retinal cell cultures, respectively.

Key words

Retina TUNEL Fluorescence microscopy Flow cytometry Tissue Adherent cell 

References

  1. 1.
    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 119:493–501PubMedCrossRefGoogle Scholar
  2. 2.
    Tanke HJ, van der Keur M (1993) Selection of defined cell types by flow-cytometric cell sorting. Trends Biotechnol 11:55–62PubMedCrossRefGoogle Scholar
  3. 3.
    Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257PubMedCrossRefGoogle Scholar
  4. 4.
    Kroemer G, Galluzzi L, Vandenabeele P et al (2009) Classification of cell death: recommendations of the nomenclature committee on cell death 2009. Cell Death Differ 16:3–11PubMedCrossRefGoogle Scholar
  5. 5.
    Doonan F, Donovan M, Gomez-Vicente V et al (2007) Bim expression indicates the pathway to retinal cell death in development and degeneration. J Neurosci 27:10887–10894PubMedCrossRefGoogle Scholar
  6. 6.
    Miller TJ, Schneider RJ, Miller JA et al (2006) Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, ci-1010, is mediated by p53-linked activation of caspase-3. Neurotoxicology 27:44–59PubMedCrossRefGoogle Scholar
  7. 7.
    Lucassen PJ, Chung WC, Vermeulen JP et al (1995) Microwave-enhanced in situ end-labeling of fragmented DNA: parametric studies in relation to postmortem delay and fixation of rat and human brain. J Histochem Cytochem 43:1163–1171PubMedCrossRefGoogle Scholar
  8. 8.
    Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H et al (1995) In situ detection of fragmented DNA (tunel assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21:1465–1468PubMedGoogle Scholar
  9. 9.
    Reme CE, Grimm C, Hafezi F et al (2000) Apoptosis in the retina: the silent death of vision. News Physiol Sci 15:120–124PubMedGoogle Scholar
  10. 10.
    Trichonas G, Murakami Y, Thanos A et al (2010) Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis. Proc Natl Acad Sci U S A 107:21695–21700PubMedCrossRefGoogle Scholar
  11. 11.
    Ebert S, Schoeberl T, Walczak Y et al (2008) Chondroitin sulfate disaccharide stimulates microglia to adopt a novel regulatory phenotype. J Leukoc Biol 84:736–740PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Tumour Biology Laboratory, Biochemistry Department, Bioscience Research InstituteUniversity College CorkCorkIreland

Personalised recommendations