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Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking

  • Florian Hoss
  • Verena Rolfes
  • Mariana R. Davanso
  • Tarcio T. Braga
  • Bernardo S. FranklinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)

Abstract

Assembly of a relatively large protein aggregate or “speck” formed by the adaptor protein ASC is a common downstream step in the activation of most inflammasomes. This unique feature of ASC allows its visualization by several imaging techniques and constitutes a reliable and feasible readout for inflammasome activation in cells and tissues. We have previously described step-by-step protocols to generate immortalized cell lines stably expressing ASC fused to a fluorescent protein for measuring inflammasome activation by confocal microscopy, and immunofluorescence of endogenous ASC in primary cells. Here, we present two more methods to detect ASC speck formation: (1) Assessment of ASC speck formation by flow cytometry; and (2) Chemical cross-linking of ASC followed by immunoblotting. These methods allow for the discrimination of inflammasome-activated versus non-activated cells, the identification of lineage-specific inflammasome activation in complex cell mixtures, and sorting of inflammasome-activated cells for further analysis.

Keywords

Innate immunity Inflammasome Inflammation Cell signaling Macrophages Flow cytometry Pulse-shape analysis 

Notes

Acknowledgments

We would like to thank Dr. Elmar Endl, and Peter Wurst from the flow cytometry core facility of the University Clinics Bonn for valuable discussion during the development of the technique and their continuous support. BF is supported by grants from the Brigitte und Dr. Konstanze Wegener-Stiftung, Germany and Internal Seed Funding Program of the Medical faculty of the University of Bonn (BONFOR).

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Florian Hoss
    • 1
  • Verena Rolfes
    • 1
  • Mariana R. Davanso
    • 1
    • 2
  • Tarcio T. Braga
    • 1
    • 2
    • 3
  • Bernardo S. Franklin
    • 1
    Email author
  1. 1.Institute of Innate Immunity, University HospitalUniversity of BonnBonnGermany
  2. 2.Department of Physiology and Biophysics, Institute of Biomedical Sciences IUniversity of São Paulo (USP)São PauloBrazil
  3. 3.Department of Immunology, Institute of Biomedical Sciences IVUniversity of São Paulo (USP)São PauloBrazil

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