Chapter title |
Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking
|
---|---|
Chapter number | 10 |
Book title |
Innate Immune Activation
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7519-8_10 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7518-1, 978-1-4939-7519-8
|
Authors |
Florian Hoss, Verena Rolfes, Mariana R. Davanso, Tarcio T. Braga, Bernardo S. Franklin |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 42 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Researcher | 9 | 21% |
Student > Ph. D. Student | 8 | 19% |
Student > Bachelor | 4 | 10% |
Student > Doctoral Student | 3 | 7% |
Student > Master | 3 | 7% |
Other | 5 | 12% |
Unknown | 10 | 24% |
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Medicine and Dentistry | 4 | 10% |
Computer Science | 1 | 2% |
Other | 2 | 5% |
Unknown | 10 | 24% |