title: A novel flow cytometric method to assess inflammasome formation.
abstract: Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; katryn.stacey@uq.edu.au d.sester@uq.edu.au.
authors.forename: David P
authors.initials: DP
authors.lastname: Sester
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia;
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Thygesen
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia;
authors.forename: Vitaliya
authors.initials: V
authors.lastname: Sagulenko
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia;
authors.forename: Parimala R
authors.initials: PR
authors.lastname: Vajjhala
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia;
authors.forename: Jasmyn A
authors.initials: JA
authors.lastname: Cridland
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia;
authors.forename: Nazarii
authors.initials: N
authors.lastname: Vitak
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; and.
authors.forename: Kaiwen W
authors.initials: KW
authors.lastname: Chen
authors.affiliation: Queensland Brain Institute, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Osborne
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; and.
authors.forename: Kate
authors.initials: K
authors.lastname: Schroder
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; and katryn.stacey@uq.edu.au d.sester@uq.edu.au.
authors.forename: Katryn J
authors.initials: KJ
authors.lastname: Stacey
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000276:immunology*; D001854:Bone Marrow Cells / Q000276:immunology; D053468:CARD Signaling Adaptor Proteins / Q000276:immunology; D020170:Caspase 1 / Q000235:genetics; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D058847:Inflammasomes / Q000032:analysis / Q000276:immunology*; D018836:Inflammation Mediators / Q000276:immunology; D008264:Macrophages / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout
chemical_list: D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D058847:Inflammasomes; D018836:Inflammation Mediators; C498534:Pycard protein, mouse; D020170:Caspase 1