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.
Similar content being viewed by others
References
Latz E, Xiao TS, Stutz A (2013) Activation and regulation of the inflammasomes. Nat Rev Immunol 13(6):397–411
Franchi L, Muñoz-Planillo R, Nuñez G (2012) Sensing and reacting to microbes through the inflammasomes. Nat Immunol 13(4):325–332
Lu A et al (2014) Unified Polymerization Mechanism for the Assemblyof ASC-Dependent Inflammasomes. Cell 156(6):1193–1206
Masumoto J et al (1999) ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J Biol Chem 274(48):33835–33838
Hoss F, Rodriguez-Alcazar JF, Latz E (2017) Assembly and regulation of ASC specks. Cell Mol Life Sci 74(7):1211–1229
Fernandes-Alnemri T et al (2007) The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14(9):1590–1604
Cai X et al (2014) Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell 156(6):1207–1222
Franklin BS et al (2014) The adaptor ASC has extracellular and “prionoid” activities that propagate inflammation. Nat Immunol 15(8):727–737
He W-T et al (2015) Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res 25(12):1285–1298
Stutz A, Horvath GL, Monks BG, Latz E (2013) ASC speck formation as a readout for inflammasome activation. Methods Mol Biol 1040:91–101
Baroja-Mazo A et al (2014) The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 15:738–748
Stutz A, Horvath GL, Monks BG, Latz E (2013) ASC speck formation as a readout for inflammasome activation. Methods in molecular biology. Humana Press, Totowa, NJ, pp 91–101
Beilharz M, De Nardo’s D, Latz E, Franklin BS (2016) Measuring NLR oligomerization II: detection of ASC speck formation by confocal microscopy and immunofluorescence. Methods Mol Biol 1417(Chapter 9):145–158
Sester DP et al (2015) A novel flow cytometric method to assess inflammasome formation. J Immunol 194(1):455–462
Ramdzan YM et al (2012) Tracking protein aggregation and mislocalization in cells with flow cytometry. Nat Methods 9(5):467–470
Arora B, Tandon R, Attri P, Bhatia R (2017) Chemical crosslinking: role in protein and peptide science. Curr Protein Pept Sci 18(9):946
Reddy N, Reddy R, Jiang Q (2015) Crosslinking biopolymers for biomedical applications. Trends Biotechnol 33(6):362–369
Mattson G et al (1993) A practical approach to crosslinking. Mol Biol Rep 17(3):167–183
Labeta MO, Fernandez N, Festenstein H (1988) Solubilisation effect of Nonidet P-40, triton X-100 and CHAPS in the detection of MHC-like glycoproteins. J Immunol Methods 112(1):133–138
Bandura DR et al (2009) Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem 81(16):6813–6822
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Hoss, F., Rolfes, V., Davanso, M.R., Braga, T.T., Franklin, B.S. (2018). Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking. In: De Nardo, D., De Nardo, C. (eds) Innate Immune Activation. Methods in Molecular Biology, vol 1714. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7519-8_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7519-8_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7518-1
Online ISBN: 978-1-4939-7519-8
eBook Packages: Springer Protocols