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Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood.
Wittmann, Nico; Behrendt, Ann-Kathrin; Mishra, Neha; Bossaller, Lukas; Meyer-Bahlburg, Almut.
  • Wittmann N; Pediatric Rheumatology, Department Pediatric and Adolescent Medicine, University Medicine, University of Greifswald, 17489 Greifswald, Germany.
  • Behrendt AK; Pediatric Rheumatology, Department Pediatric and Adolescent Medicine, University Medicine, University of Greifswald, 17489 Greifswald, Germany.
  • Mishra N; Section of Rheumatology, Department of Medicine A, University Medicine, University of Greifswald, 17489 Greifswald, Germany.
  • Bossaller L; Section of Rheumatology, Department of Medicine A, University Medicine, University of Greifswald, 17489 Greifswald, Germany.
  • Meyer-Bahlburg A; Pediatric Rheumatology, Department Pediatric and Adolescent Medicine, University Medicine, University of Greifswald, 17489 Greifswald, Germany.
Cells ; 10(11)2021 10 26.
Article in English | MEDLINE | ID: covidwho-1488493
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-1ß 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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: CARD Signaling Adaptor Proteins / Inflammasomes / Flow Cytometry Limits: Humans Language: English Year: 2021 Document Type: Article Affiliation country: Cells10112880

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Full text: Available Collection: International databases Database: MEDLINE Main subject: CARD Signaling Adaptor Proteins / Inflammasomes / Flow Cytometry Limits: Humans Language: English Year: 2021 Document Type: Article Affiliation country: Cells10112880