Therefore, we propose that accurate assessment of specks by MIFC could help guide preventive or therapeutic strategies in an array of human inflammatory diseases in which inflammasomes play an important role. By applying these tools in PBMCs from patients with distinct inflammatory disorders we demonstrate that MIFC is able to assess canonical inflammasome activation in a quantitative and statistically robust manner in clinically relevant samples. We demonstrate that this technique is also able to detect the distribution of active caspase-1 within the ASC aggregates by incubating cells with FAM-FLICA TM, a fluorochrome inhibitor of caspase-1. Here we explored the optimal imaging-based tools to measure ASC speck formation via imaging flow cytometry by using peripheral blood mononuclear cells (PBMCs) stimulated with the NLRP3 agonist Nigericin, as a positive control. Multispectral imaging flow cytometry (MIFC) combines the qualitative power of fluorescence microscopy with high throughput capabilities and multiplexing potential of flow cytometry into one single system. However, equally reliable but less time-consuming quantitative methods have emerged as a significant need in order to improve clinical assessment of inflammasome-related conditions. During this process, cytoplasmic dispersed ASC molecules cluster in one condensed micrometric-sized complex named ASC “speck,” which is traditionally assessed by fluorescence microscopy and widely accepted as a readout for canonical inflammasome activation. Once formed, this multimeric protein structure allows for the activation of caspase-1, responsible for IL-1ß/IL-18 release. Inflammasome formation requires assembly of a cytosolic sensor protein with the adapter, ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain). Canonical inflammasome activation is a tightly regulated process that has been implicated in a broad spectrum of inflammatory disorders.
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