Abstract
Inflammasome assembly results in the formation of a large intracellular protein scaffold driven by the oligomerization of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). Following inflammasome activation, ASC polymerizes to form a large singular structure termed the ASC “speck,” which is crucial for recruitment of caspase-1 and its inflammatory activity. Hence, due to the considerably large size of these structures, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide two detailed protocols for imaging ASC specks: by (1) live-cell imaging of monocyte/macrophage cell lines expressing a fluorescently tagged version of ASC and (2) immunofluorescence of endogenous ASC in cell lines and human immune cells. In addition, we outline a protocol for increasing the specificity of ASC antibodies for use in immunofluorescence.
The original version of this chapter was revised. The erratum to this chapter is available at: DOI 10.1007/978-1-4939-3289-4_19
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-4939-3566-6_19
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Acknowledgments
This work was supported by grants from the Alexander von Humboldt Foundation (B.S.F.) and the intramural BONFOR research support at the University of Bonn (B.S.F. and D.D).
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Beilharz, M., De Nardo, D., Latz, E., Franklin, B.S. (2016). Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence. In: Di Virgilio, F., Pelegrín, P. (eds) NLR Proteins. Methods in Molecular Biology, vol 1417. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3566-6_9
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DOI: https://doi.org/10.1007/978-1-4939-3566-6_9
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3566-6
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