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

Abstract

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.

Inflammatory Biomarkers of Traumatic Brain Injury

“…The inflammasome activation involves cleavage of caspase-1 through the use of a scaffolding adaptor protein known as the apoptosis-associated speck-like protein containing a caspase recruiting domain (ASC), which oligomerizes to form an ASC speck. ASC specks bind to caspase-1, resulting in the formation of the inflammasome complex [28][29][30][31] and downstream activation of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β [32].…”

Monitoring NLRP3 Inflammasome Activation and Exhaustion in Clinical Samples: A Refined Flow Cytometry Protocol for ASC Speck Formation Measurement Directly in Whole Blood after Ex Vivo Stimulation

“…Recently, after evaluation of various pre-analytical conditions (type of anticoagulant, temperature and sample storage time, etc. ), Wittman and his team provided technical recommendations regarding ASC specks detection in human PBMCs [ 16 ]. In order to facilitate clinical explorations, Cui et al reported a whole blood protocol without any subsequent ex vivo stimulation to study in vivo NLRP3 inflammasome activation level [ 17 ].…”

Inflammasome activation and formation of ASC specks in patients with juvenile idiopathic arthritis

“…The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local ethics committee (BB 014/18; BB 158/19; BB 188/20; BB032/21). After coagulation was complete, the serum was centrifuged for 10 min at 1,000 g. Peripheral blood mononuclear cells (PBMCs) were isolated from blood using density gradient centrifugation as previously described (12).…”

“…For longer storage of fixed cells, we recommend a washing step and storage of samples in PBS. Cells were stained and measured on the next day as previously described (12).…”

“…Previous studies suggest an involvement of inflammasomes in JIA pathogenesis, however cell type specific inflammasome activation has not been thoroughly investigated so far (10,11). Therefore, we recently optimized a protocol, from Sester et al (12,13), for flow cytometric detection of ASC specks ex vivo to directly determine cell type specific inflammasome activation in peripheral blood. This method is particularly suitable for pediatric patients, since only small volumes of blood can be collected.…”