title: Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence.
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.
authors.affiliation: Institute of Innate Immunity, University Hospitals, University of Bonn, Biomedizinisches Zentrum, Sigmund-Freud-Straße 25, Bonn, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Beilharz
authors.affiliation: Institute of Innate Immunity, University Hospitals, University of Bonn, Biomedizinisches Zentrum, Sigmund-Freud-Straße 25, Bonn, Germany.|Inflammation Division, Walter and Eliza Hall Institute (WEHI), 1G Royal Parade, Parkville, VIC, Australia.|Department of Medical Biology, The University of Melbourne, Parkville, 3010, Australia.
authors.forename: Dominic
authors.initials: D
authors.lastname: De Nardo
authors.affiliation: Institute of Innate Immunity, University Hospitals, University of Bonn, Biomedizinisches Zentrum, Sigmund-Freud-Straße 25, Bonn, Germany.|German Center for Neurodegenerative Diseases, Bonn, 53175, Germany.|Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, 7491, Norway.
authors.forename: Eicke
authors.initials: E
authors.lastname: Latz
authors.affiliation: Institute of Innate Immunity, University Hospitals, University of Bonn, Biomedizinisches Zentrum, Sigmund-Freud-Straße 25, Bonn, Germany. franklin@uni-bonn.de.
authors.forename: Bernardo S
authors.initials: BS
authors.lastname: Franklin
mesh_terms: D000818:Animals; D053468:CARD Signaling Adaptor Proteins / Q000737:chemistry*; D020170:Caspase 1 / Q000378:metabolism; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D055503:Protein Multimerization; D000074084:THP-1 Cells
chemical_list: D053468:CARD Signaling Adaptor Proteins; D058847:Inflammasomes; C402202:PYCARD protein, human; C498534:Pycard protein, mouse; D020170:Caspase 1
keywords: ASC; Confocal microscopy; Flow cytometry; Immunofluorescence; Inflammasome; Live-cell imaging; Speck