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The Inflammasome pp 65–84Cite as

Measuring Inflammasome Activation in Response to Bacterial Infection

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1040))

Abstract

Inflammasomes are multi-protein signaling platforms assembled in response to viral and bacterial pathogens as well as endogenous danger signals. Inflammasomes serve as activation platforms for the mammalian cysteine protease caspase-1, a central mediator of innate immunity. The hallmarks of inflammasome activation are the processing of caspase-1, the maturation and release of interleukin-1β (IL-1β) and the induction of pyroptosis, a lytic inflammatory cell death. This protocol describes methods for studying inflammasome activation in response to bacterial pathogens in bone-marrow derived murine macrophages (BMDMs). In particular, we outline the protocols to measure cytokine maturation by ELISA and pyroptosis by the release of Lactate Dehydrogenase (LDH). In addition, we describe methods to visualize endogenous ASC specks or foci in infected cells and to study the release of processed caspase-1, caspase-11 and mature cytokines into the cell supernatant by Western blotting. General considerations are discussed to design and optimize the infection protocol for the study of inflammasome activation by other bacterial pathogens.

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Acknowledgment

We would like to thank Maikke B. Ohlson and Jens Kortmann for critical reading of the manuscript. This work was supported by awards AI095396 and AI08972 from the National Institute of Allergy and Infectious Diseases to D.M., a Stanford Digestive Disease Center (DDC) pilot grant to P.B. and a long-term fellowship from the Human Frontiers in Science Program (HFSP) to P.B.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA

    Petr Broz & Denise M. Monack

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  1. Petr Broz
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  2. Denise M. Monack
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Editors and Affiliations

  1. , Institute of Innate Immunity, University of Bonn, Sigmund-Freud-Strasse 25, Bonn, 53105, Germany

    Christine M. De Nardo

  2. , Institute of Innate Immunity, University of Bonn, Sigmund-Freud-Str. 25, Bonn, 53105, Germany

    Eicke Latz

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Broz, P., Monack, D.M. (2013). Measuring Inflammasome Activation in Response to Bacterial Infection. In: De Nardo, C., Latz, E. (eds) The Inflammasome. Methods in Molecular Biology, vol 1040. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-523-1_6

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  • DOI: https://doi.org/10.1007/978-1-62703-523-1_6

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-522-4

  • Online ISBN: 978-1-62703-523-1

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