Broz Petr, Monack Denise M
Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA.
Methods Mol Biol. 2013;1040:65-84. doi: 10.1007/978-1-62703-523-1_6.
Inflammasomes are multi-protein signaling platforms assembled in response to viral and bacterial pathogens as well as endogenous danger signals. Inflammasomes serve as activation platforms for the mammalian cysteine protease caspase-1, a central mediator of innate immunity. The hallmarks of inflammasome activation are the processing of caspase-1, the maturation and release of interleukin-1β (IL-1β) and the induction of pyroptosis, a lytic inflammatory cell death. This protocol describes methods for studying inflammasome activation in response to bacterial pathogens in bone-marrow derived murine macrophages (BMDMs). In particular, we outline the protocols to measure cytokine maturation by ELISA and pyroptosis by the release of Lactate Dehydrogenase (LDH). In addition, we describe methods to visualize endogenous ASC specks or foci in infected cells and to study the release of processed caspase-1, caspase-11 and mature cytokines into the cell supernatant by Western blotting. General considerations are discussed to design and optimize the infection protocol for the study of inflammasome activation by other bacterial pathogens.
炎性小体是多蛋白信号平台，其组装是对病毒和细菌病原体以及内源性危险信号作出的反应。炎性小体作为哺乳动物半胱氨酸蛋白酶caspase-1的激活平台，caspase-1是先天免疫的核心介质。炎性小体激活的标志是caspase-1的加工、白细胞介素-1β（IL-1β）的成熟和释放以及细胞焦亡的诱导，细胞焦亡是一种溶解性炎性细胞死亡。本方案描述了在骨髓来源的小鼠巨噬细胞（BMDM）中研究炎性小体对细菌病原体激活反应的方法。特别地，我们概述了通过酶联免疫吸附测定（ELISA）测量细胞因子成熟以及通过乳酸脱氢酶（LDH）释放测量细胞焦亡的方案。此外，我们描述了在感染细胞中可视化内源性凋亡相关斑点样蛋白（ASC）斑点或病灶以及通过蛋白质免疫印迹法研究加工后的caspase-1、caspase-11和成熟细胞因子释放到细胞上清液中的方法。讨论了用于设计和优化其他细菌病原体激活炎性小体研究的感染方案的一般注意事项。
