Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood

Inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multiprotein complex, known as the ASC speck. Redistribution of cytosolic ASC to this complex has been widely used as a readout for inflammasome activation and precedes the downstream proteolytic release of the pr...

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Autores principales:	Nico Wittmann, Ann-Kathrin Behrendt, Neha Mishra, Lukas Bossaller, Almut Meyer-Bahlburg
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	inflammasome ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) flow cytometry human PBMC clinical study Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/d88e83223c05471d9d77568b3e1df41c
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spelling	oai:doaj.org-article:d88e83223c05471d9d77568b3e1df41c2021-11-25T17:08:30ZInstructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood10.3390/cells101128802073-4409https://doaj.org/article/d88e83223c05471d9d77568b3e1df41c2021-10-01T00:00:00Zhttps://www.mdpi.com/2073-4409/10/11/2880https://doaj.org/toc/2073-4409Inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multiprotein complex, known as the ASC speck. Redistribution of cytosolic ASC to this complex has been widely used as a readout for inflammasome activation and precedes the downstream proteolytic release of the proinflammatory cytokines, IL-1β and IL-18. Although inflammasomes are important for many diseases such as periodic fever syndromes, COVID-19, gout, sepsis, atherosclerosis and Alzheimer’s disease, only a little knowledge exists on the precise and cell type specific occurrence of inflammasome activation in patient samples ex vivo. In this report, we provide detailed information about the optimal conditions to reliably identify inflammasome activated monocytes by ASC speck formation using a modified flow cytometric method introduced by Sester et al. in 2015. Since no protocol for optimal sample processing exists, we tested human blood samples for various conditions including anticoagulant, time and temperature, the effect of one freeze–thaw cycle for PBMC storage, and the fast generation of a positive control. We believe that this flow cytometric protocol will help researchers to perform high quality translational research in multicenter studies, and therefore provide a basis for investigating the role of the inflammasome in the pathogenesis of various diseases.Nico WittmannAnn-Kathrin BehrendtNeha MishraLukas BossallerAlmut Meyer-BahlburgMDPI AGarticleinflammasomeASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain)flow cytometryhumanPBMCclinical studyBiology (General)QH301-705.5ENCells, Vol 10, Iss 2880, p 2880 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	inflammasome ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) flow cytometry human PBMC clinical study Biology (General) QH301-705.5
spellingShingle	inflammasome ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) flow cytometry human PBMC clinical study Biology (General) QH301-705.5 Nico Wittmann Ann-Kathrin Behrendt Neha Mishra Lukas Bossaller Almut Meyer-Bahlburg Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood
description	Inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multiprotein complex, known as the ASC speck. Redistribution of cytosolic ASC to this complex has been widely used as a readout for inflammasome activation and precedes the downstream proteolytic release of the proinflammatory cytokines, IL-1β and IL-18. Although inflammasomes are important for many diseases such as periodic fever syndromes, COVID-19, gout, sepsis, atherosclerosis and Alzheimer’s disease, only a little knowledge exists on the precise and cell type specific occurrence of inflammasome activation in patient samples ex vivo. In this report, we provide detailed information about the optimal conditions to reliably identify inflammasome activated monocytes by ASC speck formation using a modified flow cytometric method introduced by Sester et al. in 2015. Since no protocol for optimal sample processing exists, we tested human blood samples for various conditions including anticoagulant, time and temperature, the effect of one freeze–thaw cycle for PBMC storage, and the fast generation of a positive control. We believe that this flow cytometric protocol will help researchers to perform high quality translational research in multicenter studies, and therefore provide a basis for investigating the role of the inflammasome in the pathogenesis of various diseases.
format	article
author	Nico Wittmann Ann-Kathrin Behrendt Neha Mishra Lukas Bossaller Almut Meyer-Bahlburg
author_facet	Nico Wittmann Ann-Kathrin Behrendt Neha Mishra Lukas Bossaller Almut Meyer-Bahlburg
author_sort	Nico Wittmann
title	Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood
title_short	Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood
title_full	Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood
title_fullStr	Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood
title_full_unstemmed	Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood
title_sort	instructions for flow cytometric detection of asc specks as a readout of inflammasome activation in human blood
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/d88e83223c05471d9d77568b3e1df41c
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Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood

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