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Induction and Detection of Necroptotic Cell Death in Mammalian Cell Culture

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Detection of Cell Death Mechanisms

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2255))

Abstract

The study of necroptosis is a rapidly growing field in current research of cell death mechanisms and cancer treatment strategies. While apoptotic cells can be reliably identified via annexin V assay, necroptosis is not associated with exposure of easily detectable markers. The most reliable way to identify necroptotic events is immunochemical detection of active phosphorylated RIPK1, RIPK3, and MLKL proteins facilitating necroptosis execution. This chapter describes a detailed protocol on necroptosis induction in human colon adenocarcinoma HT-29 cells, preparation of various positive and negative controls, detection of necroptosis mediator proteins via Western Blot analysis, and interpretation of results. This protocol allows reliable and specific detection of necroptosis in cell culture or tissue samples, and it provides a well-established model suitable for detailed studies of necroptosis molecular mechanisms in vitro.

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Authors and Affiliations

  1. The Hormel Institute, University of Minnesota, Austin, MN, USA

    Mikhail Chesnokov, Imran Khan & Ilana Chefetz

  2. Masonic Cancer Center, Minneapolis, MN, USA

    Ilana Chefetz

  3. Stem Cell Institute, Minneapolis, MN, USA

    Ilana Chefetz

  4. Department of Obstetrics, Gynecology and Women’s Health, Minneapolis, MN, USA

    Ilana Chefetz

Authors
  1. Mikhail Chesnokov
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  2. Imran Khan
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  3. Ilana Chefetz
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Corresponding author

Correspondence to Ilana Chefetz .

Editor information

Editors and Affiliations

  1. Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA

    Ayesha B. Alvero

  2. Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA

    Gil G. Mor

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Chesnokov, M., Khan, I., Chefetz, I. (2021). Induction and Detection of Necroptotic Cell Death in Mammalian Cell Culture. In: Alvero, A.B., Mor, G.G. (eds) Detection of Cell Death Mechanisms. Methods in Molecular Biology, vol 2255. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1162-3_11

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  • DOI: https://doi.org/10.1007/978-1-0716-1162-3_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1161-6

  • Online ISBN: 978-1-0716-1162-3

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