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...

Full description

Saved in:
Bibliographic Details
Published in:Cells (Basel, Switzerland) Switzerland), 2021-10, Vol.10 (11), p.2880
Main Authors: Wittmann, Nico, Behrendt, Ann-Kathrin, Mishra, Neha, Bossaller, Lukas, Meyer-Bahlburg, Almut
Format: Article
Language:English
Subjects:
Adapter proteins
Alzheimer's disease
Antibodies
Arteriosclerosis
ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain)
Cell culture
clinical study
COVID-19
Cytokines
Fever
Flow cytometry
Freeze-thawing
Gout
human
IL-1β
inflammasome
Inflammasomes
Inflammation
Interleukin 18
Microscopy
Monocytes
Neurodegenerative diseases
PBMC
Peripheral blood mononuclear cells
Proteolysis
Reagents
Sepsis
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:2073-4409
2073-4409
DOI:10.3390/cells10112880