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. 2019 Apr 18;4(8):e127113. doi: 10.1172/jci.insight.127113

Figure 7. Quantification of canonical inflammasome activation in CD14+ monocytes.

Figure 7

PBMCs from IR (n = 5), INR (n = 5), EXID2, EXID4, and a healthy subject were incubated with a fluorochrome-labeled inhibitor of caspase-1 (FAM-FLICA), stained for monocyte identification with antibodies against CD14 and the intracellular apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), and evaluated by imaging flow cytometry. (A) The number of monocytes showing spontaneous ASC speck formation was quantified after application of the feature Area Threshold versus Modulation Morphology in the CD14-expressing monocyte gate, by ImageStream Data Exploration and Analysis Software (IDEAS 6.2.64.0, MilliporeSigma). Each EXID subject is identified by a different gray-filled shape. *P ≤ 0.05 in the comparison indicated by the black horizontal line as determined by Mann-Whitney U test. (B) Representative images showing colocalization of active caspase-1 with ASC specks were selected after Bright Detail Similarity was applied in the ASC speck gate. Images show, respectively: brightfield (BF), ASC and FLICA fluorescence followed by a composite image containing BF, and the fluorescence of ASC and FLICA merged.