Abstract
The detection of pathogen- or danger-associated molecular patterns during an inflammatory injury triggers the activation of cytosolic sensors known as inflammasomes. Once stimulated, these protein complexes can connect to the adaptor protein ASC, which in turn recruits the effector enzyme caspase-1, forming a polymeric structure known as ASC speck. This protein scaffold is responsible for processing cytokines of the IL-1 family into their active forms and evoking the cleavage of gasdermin D, ultimately leading to cell death by pyroptosis. Due to its micrometric size, the specks are used as a readout for inflammasome activation and for the better comprehension of this important immune pathway. In this chapter, a detailed protocol is presented for the study of the formation of inflammasome specks in living cells using confocal microscopy.
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Acknowledgments
I would like to thank Dr. Gabor Horvath, from the Microscopy Core Facility of the University of Bonn, for the fundamental assistance for the live imaging of ASC specking, and Dr. Damien Bertheloot for providing the THP-1 ASC-TagGFP2 reporter cell line.
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After the addition of nigericin to LPS-primed THP-1 ASC-TagGFP2 cells, Z-stacks of 20 μm (1 μm-layers) were imaged for 60 min in cycles of 1 min, resulting in 60 individual frames made from the maximum projection of the layers. The frames were analyzed and exported as an animation of 10 frames/second using the software Imaris 9.1.2 (MP4 9831 kb)
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Secchim Ribeiro, L. (2022). Imaging of Inflammasome Speck Formation in Living Cells. In: Abdul-Sater, A.A. (eds) The Inflammasome. Methods in Molecular Biology, vol 2459. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2144-8_16
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DOI: https://doi.org/10.1007/978-1-0716-2144-8_16
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