Wu Yung-Hsuan, Lai Ming-Zong
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan.
Institute of Immunology, National Taiwan University, Taipei, Taiwan.
Methods Mol Biol. 2016;1417:185-95. doi: 10.1007/978-1-4939-3566-6_12.
The NLRP3 inflammasome is assembled in macrophages and monocytes in response to inflammatory and danger stimuli. The atypical nature of the NLRP3 complex impedes detection of NLRP3 inflammasome formation by conventional biochemical and cell biology methods. In situ proximity ligation assay (PLA) provides an alternative method of detection, localization, and quantification of protein-protein interactions in tissue and cell samples. Two primary antibodies raised in different species detect the two proteins of interest. When the proteins are in close proximity, secondary antibodies conjugated with specific DNA probes hybridize with linking oligonucleotides to form a DNA bridge between the two proteins. Amplification of the DNA bridge then facilitates detection by microscopy using fluorescence probes. Here, we describe application of in situ PLA to detect NLRP3 inflammasome assembly in mouse bone marrow-derived macrophages and human monocyte cell line THP1.
NLRP3炎性小体在巨噬细胞和单核细胞中组装，以响应炎症和危险刺激。NLRP3复合物的非典型性质阻碍了通过传统生化和细胞生物学方法检测NLRP3炎性小体的形成。原位邻近连接分析（PLA）提供了一种在组织和细胞样本中检测、定位和定量蛋白质-蛋白质相互作用的替代方法。在不同物种中产生的两种一抗检测两种感兴趣的蛋白质。当蛋白质紧密相邻时，与特定DNA探针偶联的二抗与连接寡核苷酸杂交，在两种蛋白质之间形成DNA桥。然后，DNA桥的扩增便于使用荧光探针通过显微镜进行检测。在这里，我们描述了原位PLA在检测小鼠骨髓来源的巨噬细胞和人单核细胞系THP1中NLRP3炎性小体组装方面的应用。
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