测量NLR寡聚化II：通过共聚焦显微镜和免疫荧光检测ASC斑点形成。

Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence.

作者信息

Beilharz Michael, De Nardo Dominic, Latz Eicke, Franklin Bernardo S

机构信息

Institute of Innate Immunity, University Hospitals, University of Bonn, Biomedizinisches Zentrum, Sigmund-Freud-Straße 25, Bonn, Germany.

Inflammation Division, Walter and Eliza Hall Institute (WEHI), 1G Royal Parade, Parkville, VIC, Australia.

出版信息

Methods Mol Biol. 2016;1417:145-58. doi: 10.1007/978-1-4939-3566-6_9.

DOI:10.1007/978-1-4939-3566-6_9

PMID:27221487

Abstract

Inflammasome assembly results in the formation of a large intracellular protein scaffold driven by the oligomerization of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). Following inflammasome activation, ASC polymerizes to form a large singular structure termed the ASC "speck," which is crucial for recruitment of caspase-1 and its inflammatory activity. Hence, due to the considerably large size of these structures, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide two detailed protocols for imaging ASC specks: by (1) live-cell imaging of monocyte/macrophage cell lines expressing a fluorescently tagged version of ASC and (2) immunofluorescence of endogenous ASC in cell lines and human immune cells. In addition, we outline a protocol for increasing the specificity of ASC antibodies for use in immunofluorescence.

摘要

炎性小体组装导致由含CARD结构域的凋亡相关斑点样蛋白（ASC）寡聚驱动形成一个大型细胞内蛋白质支架。炎性小体激活后，ASC聚合形成一个称为ASC“斑点”的大型单一结构，这对于半胱天冬酶-1的募集及其炎症活性至关重要。因此，由于这些结构尺寸相当大，通过显微镜很容易将ASC斑点作为炎性小体激活的简单上游读数进行观察。在这里，我们提供了两种用于成像ASC斑点的详细方案：（1）对表达荧光标记ASC的单核细胞/巨噬细胞系进行活细胞成像，以及（2）对细胞系和人类免疫细胞中的内源性ASC进行免疫荧光检测。此外，我们概述了一种提高用于免疫荧光的ASC抗体特异性的方案。