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 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.

Download Full-text

A flow cytometric method for determining the binding of coagulation factor X to monocytes in whole human blood

Journal of Immunological Methods ◽

10.1016/j.jim.2004.03.016 ◽

2004 ◽

Vol 292 (1-2) ◽

pp. 207-215 ◽

Cited By ~ 1

Author(s):

Alexandre Le Guyader ◽

Grace Davis-Gorman ◽

Jack G. Copeland ◽

Paul F. McDonagh

Keyword(s):

Human Blood ◽

Coagulation Factor ◽

Factor X ◽

Flow Cytometric Method ◽

Flow Cytometric

Download Full-text

Flow Cytometric Detection of Activated Platelets in Pregnant Women Prior to the Development of Pre-Eclampsia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649882 ◽

1995 ◽

Vol 74 (04) ◽

pp. 1059-1063 ◽

Cited By ~ 59

Author(s):

Sarah L Janes ◽

Phillipa M Kyle ◽

Christopher Redman ◽

Alison H Goodall

Keyword(s):

Blood Flow ◽

Pregnant Women ◽

Whole Blood ◽

Ex Vivo ◽

Critical Role ◽

Antigen Expression ◽

Normal Pregnancy ◽

Flow Cytometric Method ◽

Flow Cytometric ◽

Activated Platelets

SummaryPre-eclampsia is a common complication of pregnancy, in which platelets may have an early pathogenetic role. In this prospective study a whole blood flow cytometric method has been used to detect circulating activated platelets in pregnant women prior to the development of pre-eclampsia. Activated platelets were identified by bound fibrinogen or by CD63 antigen expression. Of 121 healthy primiparous women studied at 28 weeks of pregnancy, 18 (15%) developed clinical pre-eclampsia six to thirteen weeks later. The platelets of these women showed increased fibrinogen binding ex vivo (5.1% platelets positive, compared with 3.4% in those who completed a normal pregnancy, p <0.02), and increased CD63 antigen expression (0.73% positive compared to 0.45%, p = 0.01). In contrast, no differences between the women with different outcomes were detected at 28 weeks in platelet counts, or plasma ß-thromboglobulin levels. These findings confirm that whole blood flow cytometry is a sensitive technique for investigating platelet activation in a clinical setting and support the hypothesis that platelets have a critical role in the pathogenesis of pre-eclampsia

Download Full-text

THU0019 INTRON REGIONS OF PIK3AP1 (BCAP) AND SPON2 (SPONDIN-2) GENES ARE DIFFERENTIALLY METHYLATED IN PATIENTS WITH PERIODIC FEVER, APHTHOUS STOMATITIS, PHARYNGITIS AND ADENITIS (PFAPA) SYNDROME

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5512 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 223.3-223

Author(s):

E. Lovšin ◽

J. Kovac ◽

T. Tesovnik ◽

N. Toplak ◽

D. Perko ◽

...

Keyword(s):

Dna Methylation ◽

Pattern Recognition ◽

Periodic Fever ◽

Aphthous Stomatitis ◽

Extracellular Matrix Protein ◽

Inflammasome Activation ◽

Cervical Adenitis ◽

Pfapa Syndrome ◽

Pattern Recognition Molecule ◽

Recognition Molecule

Background:Periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome is the most common autoinflammatory disease in children, often grouped together with hereditary periodic fever syndromes, although its cause and hereditary nature remain unexplained.Objectives:We investigated whether a differential DNA methylation was present in DNA from peripheral blood mononuclear cells (PBMC) in patients with PFAPA versus a group of healthy young individuals.Methods:A whole epigenome analysis (MeDIP and MBD) was performed using pooled DNA libraries enriched for methylated genomic regions. Of identified candidate genes, two with most significantly different methylation leves were further evaluated with methylation specific restriction enzymes coupled with qPCR (MSRE-qPCR).Results:The analysis showed thatPIK3AP1andSPON2intronic gene regions are differentially methylated in patients with PFAPA. MSRE-qPCR proved as a quick, reliable and cost-effective method to confirm results from MeDIP and MBD.Conclusion:Our findings indicate that B cell adapter protein (BCAP) as PI3K binding inhibitor of inflammation and spondin-2 (SPON2) as a pattern recognition molecule and integrin ligand could play a role in etiology of PFAPA. Their role and impact of changed DNA methylation in PFAPA etiology and autoinflammation need further investigation.References:[1]Wekell P. Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome – PFAPA syndrome. Press Medicale [Internet]. 2019;48(1):e77–87. Available from:https://doi.org/10.1016/j.lpm.2018.08.016[2]K. Theodoropoulou, F. Vanoni, and M. Hofer, “Periodic Fever, Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis (PFAPA) Syndrome: a Review of the Pathogenesis,”Curr. Rheumatol. Rep., vol. 18:18, 2016.[3]Carpentier SJ, Ni M, Duggan JM, James RG, Cookson BT, Hamerman JA. The signaling adaptor BCAP inhibits NLRP3 and NLRC4 inflammasome activation in macrophages through interactions with Flightless-1. Sci Signal. 2019;12(581).[4]He YW, Li H, Zhang J, Hsu CL, Lin E, Zhang N, et al. The extracellular matrix protein mindin is a pattern-recognition molecule for microbial pathogens. Nat Immunol. 2004;5(1):88–97.Disclosure of Interests:None declared

Download Full-text