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- W4313360058 abstract "Abstract NLRP proteins interact with the adaptor ASC to recruit and activate caspase-1-dependent maturation of IL-1β and IL-18 in a multiprotein ‘inflammasome’ complex. Inflammasome activation occurs in response to structurally diverse agonists triggering NLRP activation leading to rapid localization of NLRP:ASC complexes in a typically singular perinuclear ‘speck’. At present, formation of inflammasome specks can be assayed by microscopy, which is time consuming, or a flow cytometric analysis technique evaluating altered width and height of ASC pulse (TOFIE), which provides no visualization of the speck. We have developed a technique utilizing the advantages of imaging flow cytometry that combines high-throughput sample collection and high magnification (60×) microscopic visualization. This technique not only detects the presence of ASC specks but also determines the distribution of active caspase-1 within the cell using the fluorescent caspase-1 substrate FLICA. This assay allows for high-throughput imaging of ASC-containing specks and characterization of these specks in terms of number and area. Additionally, this technique also determines the location of active caspase-1 and enumerates aggregates of active caspase-1. This method is more sensitive than the present flow cytometric method, TOFIE, which fails to differentiate between active inflammasome specks and false positive signals due to accumulation/aggregation of ASC. The capacity of this method to detect and quantify native as well as reconstituted inflammasome specks with associated caspase-1 activity renders it more powerful for quantifying the impact of mutations on the molecular interaction of NLRP proteins with ASC and accompanying caspase-1 activation." @default.
- W4313360058 created "2023-01-06" @default.
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- W4313360058 date "2017-05-01" @default.
- W4313360058 modified "2023-09-25" @default.
- W4313360058 title "A novel method of assessing ASC specks and caspase-1 activity by imaging flow cytometry" @default.
- W4313360058 doi "https://doi.org/10.4049/jimmunol.198.supp.64.2" @default.
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