Stutz Andrea, Horvath Gabor L, Monks Brian G, Latz Eicke
Institute of Innate Immunity, University Hospital, University of Bonn, Bonn, Germany.
Methods Mol Biol. 2013;1040:91-101. doi: 10.1007/978-1-62703-523-1_8.
All inflammasomes require the adapter protein apoptosis associated speck-like protein containing a CARD (ASC) for the activation of caspase-1. After inflammasome activation, ASC assembles into a large protein complex, which is termed "speck". ASC specks can be observed as they reach a size of around 1 μm and in most cells only one speck forms upon inflammasome activation. Hence, ASC speck formation can be used as a simple upstream readout for inflammasome activation. Here, we describe a method for analyzing inflammasome activation by ASC speck visualization. First, we describe the generation of a clonal inflammasome reporter macrophage cell line overexpressing fluorescently tagged ASC. We then discuss stimulation conditions and the microscopic evaluation of ASC speck formation.
所有炎性小体都需要衔接蛋白含半胱天冬酶激活和招募结构域的凋亡相关斑点样蛋白（ASC）来激活半胱天冬酶-1。炎性小体激活后，ASC组装成一个大的蛋白质复合物，称为“斑点”。当ASC斑点达到约1μm大小时就可以观察到，并且在大多数细胞中，炎性小体激活时仅形成一个斑点。因此，ASC斑点形成可作为炎性小体激活的一个简单的上游读数。在此，我们描述一种通过ASC斑点可视化来分析炎性小体激活的方法。首先，我们描述过表达荧光标记ASC的克隆炎性小体报告巨噬细胞系的生成。然后，我们讨论刺激条件以及ASC斑点形成的显微镜评估。
