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. 2021 Sep 28;12:720655. doi: 10.3389/fimmu.2021.720655

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Copyright © 2021 Diaz-del-Olmo, Worboys, Martin-Sanchez, Gritsenko, Ambrose, Tannahill, Nichols, Lopez-Castejon and Davis

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The MAC triggers inflammasome assembly and localizes to the NLRP3-ASC speck. (A–F) Human MDMs (A–E) and THP1^nlrp3-/-/eGFP-nlrp3 cells (F) were treated with vehicles or primed with 1 µg/mL LPS for 3 hrs followed by stimulation with 10 µg/mL anti-CD59 mAb, C5b6, C7, C8 and C9 labelled with JF-549 (MAC) for the indicated time (A–C) or 3hrs (D–F). (A) Representative confocal images of C9 (red) and ASC (cyan) overtime. (B) Percentage of cells with ASC specks over time. (C) Percentage of cells with C9 over time. (D) Confocal imaging of C9 (red), ASC (cyan) and TCC (green). (E) Percentage of ASC speck positive cells with C9 in the speck. (F) Confocal imaging of C9 (red), ASC (cyan) and NLRP3 (green). (A, D, F) Dashed lines represent the outline of the cell, arrows point to specks, magenta lines correspond to the plotted line profiles and scale bars are 10 µm (A) and 5 µm (D, F). (B, C, E) Data is plotted as mean ± SD, each color represents a matched donor. (B, C) Statistical significance was measured by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (A–F) Data is representative of 3 independent experiments.