This provides rapid quantification of responding cell number and correlation with the expression level of inflammasome components within single cells. A novel flow cytometric method to assess inflammasome formation. Journal of Immunology 1 Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain ASC into a single speck.
We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.
Measuring the inflammasome. Pubmed Gross O Inflammasomes are multiprotein complexes whose activity has been implicated in physiological and pathological inflammation. This protocol covers the methods required to study inflammasome activation using mouse bone marrow-derived dendritic cells BMDCs as a model system.
These methods can be useful for the study of potential inflammasome activators, and of the signaling pathways involved in inflammasome activation. General considerations are provided that may help in the design and optimization of modified methods for the study of other types of inflammasomes and in other cell types. Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages. Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species.
Monocytes phenotypically change during stimulation over time. Fig 6.
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Supporting information. S1 Fig. Gating strategies for flow cytometry analysis. S2 Fig.
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S3 Fig. S4 Fig. S5 Fig.
S6 Fig. Baseline values for monocyte subpopulations. S7 Fig. Dynamics and kinetics of distribution of monocytic subsets during inflammasome activation. S8 Fig. S9 Fig.
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References 1. Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol. ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation. Cell Death Dis. A protective role for inflammasome activation following injury. Regulation of inflammasome signaling. Nature immunology. Journal of immunology Baltimore, Md: FEBS letters. Modulatory mechanisms controlling the NLRP3 inflammasome in inflammation: recent developments.
Current opinion in immunology. Mechanism of NLRP3 inflammasome activation. Annals of the New York Academy of Sciences. The inflammasome: a caspaseactivation platform that regulates immune responses and disease pathogenesis. Activation and regulation of the inflammasomes. Nature reviews Immunology. Caspase-4 is required for activation of inflammasomes. A critical role for human caspase-4 in endotoxin sensitivity. Science New York, NY. FADD and caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes.
Interleukin and IL binding protein. Frontiers in immunology. Sustained effects of interleukin-1 receptor antagonist treatment in type 2 diabetes. Diabetes care.
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NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Journal of leukocyte biology. Boraschi D, Dinarello CA. IL in autoimmunity: review. European cytokine network.
Caspase-Glo® 1 Inflammasome Assay
Elevation of plasma interleukin concentration is correlated with disease activity in systemic lupus erythematosus. Rheumatology Oxford, England. Applying caspase-1 inhibitors for inflammasome assays in human whole blood. Journal of immunological methods. Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages. ATP-stimulated release of interleukin IL -1beta and IL requires priming by lipopolysaccharide and is independent of caspase-1 cleavage.
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Buy eBook. Buy Hardcover. FAQ Policy. About this book This book describes current methods for the identification and characterization of the major hallmarks of senescent cells.