A novel flow cytometric method to assess inflammasome formation

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-assoc...

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Published in	The Journal of immunology (1950) Vol. 194; no. 1; pp. 455 - 462
Main Authors	Sester, David P, Thygesen, Sara J, Sagulenko, Vitaliya, Vajjhala, Parimala R, Cridland, Jasmyn A, Vitak, Nazarii, Chen, Kaiwen W, Osborne, Geoffrey W, Schroder, Kate, Stacey, Katryn J
Format	Journal Article
Language	English
Published	United States 01.01.2015
Subjects	Animals Apoptosis - immunology Apoptosis Regulatory Proteins - genetics Apoptosis Regulatory Proteins - immunology Bone Marrow Cells - immunology CARD Signaling Adaptor Proteins - immunology Caspase 1 - genetics Cell Line Flow Cytometry - methods HEK293 Cells Humans Inflammasomes - analysis Inflammasomes - immunology Inflammation Mediators - immunology Macrophages - immunology Mice Mice, Knockout
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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.
AbstractList	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.
Author	Stacey, Katryn J Sester, David P Vajjhala, Parimala R Cridland, Jasmyn A Schroder, Kate Chen, Kaiwen W Vitak, Nazarii Thygesen, Sara J Sagulenko, Vitaliya Osborne, Geoffrey W
Author_xml	– sequence: 1 givenname: David P surname: Sester fullname: Sester, David P email: katryn.stacey@uq.edu.au, d.sester@uq.edu.au organization: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; katryn.stacey@uq.edu.au d.sester@uq.edu.au – sequence: 2 givenname: Sara J surname: Thygesen fullname: Thygesen, Sara J organization: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia – sequence: 3 givenname: Vitaliya surname: Sagulenko fullname: Sagulenko, Vitaliya organization: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia – sequence: 4 givenname: Parimala R surname: Vajjhala fullname: Vajjhala, Parimala R organization: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia – sequence: 5 givenname: Jasmyn A surname: Cridland fullname: Cridland, Jasmyn A organization: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia – sequence: 6 givenname: Nazarii surname: Vitak fullname: Vitak, Nazarii organization: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia – sequence: 7 givenname: Kaiwen W surname: Chen fullname: Chen, Kaiwen W organization: Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; and – sequence: 8 givenname: Geoffrey W surname: Osborne fullname: Osborne, Geoffrey W organization: Queensland Brain Institute, University of Queensland, Brisbane, Queensland 4072, Australia – sequence: 9 givenname: Kate surname: Schroder fullname: Schroder, Kate organization: Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; and – sequence: 10 givenname: Katryn J surname: Stacey fullname: Stacey, Katryn J email: katryn.stacey@uq.edu.au, d.sester@uq.edu.au organization: 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
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SubjectTerms	Animals Apoptosis - immunology Apoptosis Regulatory Proteins - genetics Apoptosis Regulatory Proteins - immunology Bone Marrow Cells - immunology CARD Signaling Adaptor Proteins - immunology Caspase 1 - genetics Cell Line Flow Cytometry - methods HEK293 Cells Humans Inflammasomes - analysis Inflammasomes - immunology Inflammation Mediators - immunology Macrophages - immunology Mice Mice, Knockout
Title	A novel flow cytometric method to assess inflammasome formation
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