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In Situ Detection of DNA Damage pp 289–299Cite as

Detection of Caspases Activation In Situ by Fluorochrome-Labeled Inhibitors of Caspases (FLICA)

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 203))

Abstract

Caspases are cysteine-aspartic acid specific proteases that are activated in response to different cell death inducing stimuli (1–3). Their activation initiates specific cleavage of the respective target proteins and therefore is considered to be a marker of the irreversible steps that lead to cell demise (reviews 4,5). Caspases specifically recognize a four-amino acid sequence on their substrate proteins; the carboxyl end of aspartic acid within this sequence is the target for cleavage. Several approaches have been developed to detect the process of caspases activation. Because the activation involves the transcatalytic cleavage of the zymogen pro-caspases (reviews 6–8) the cleavage products having lower molecular weight than the zymogen can be revealed electrophoretically and identified in Western blots using caspase-specific antibodies. Another approach utilizes the fluorogenic (or chromogenic) substrates of caspases. Peptide substrates were developed which are colorless or nonfluorescent, but upon cleavage, generate colored or fluorescing products (9–11). Utility of these two approaches, however, was limited to the measurement of caspases activation in cell extracts thereby providing no information on the in situ caspases activation. This would allow one to study individual cells, assess heterogeneity of cell populations, or reveal correlation with other cell attributes.

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Author information

Authors and Affiliations

  1. Brander Cancer Research Institute, New York Medical College, Valhalla, NY

    Zbigniew Darzynkiewicz, Elzbieta Bedner & Piotr Smolewski

  2. Immunochemistry Technologies, Bloomington, MN

    Brian W. Lee & Gary L. Johnson

Authors
  1. Zbigniew Darzynkiewicz
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  2. Elzbieta Bedner
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  3. Piotr Smolewski
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  4. Brian W. Lee
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  5. Gary L. Johnson
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Editor information

Editors and Affiliations

  1. Baylor College of Medicine, Houston, TX

    Vladimir V. Didenko

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© 2002 Humana Press Inc.

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Darzynkiewicz, Z., Bedner, E., Smolewski, P., Lee, B.W., Johnson, G.L. (2002). Detection of Caspases Activation In Situ by Fluorochrome-Labeled Inhibitors of Caspases (FLICA). In: Didenko, V.V. (eds) In Situ Detection of DNA Damage. Methods in Molecular Biology, vol 203. Humana Press. https://doi.org/10.1385/1-59259-179-5:289

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  • DOI: https://doi.org/10.1385/1-59259-179-5:289

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-952-0

  • Online ISBN: 978-1-59259-179-4

  • eBook Packages: Springer Protocols

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