Zhang Ping, Culver-Cochran Ashley, Stevens Stanley M, Liu Bin
Department of Pharmacodynamics, College of Pharmacy, University of Florida, 1345 Center Drive, Box 100487, Gainesville, FL, 32610, USA.
Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, ISA 2015, Tampa, FL, 33620, USA.
Methods Mol Biol. 2017;1598:285-293. doi: 10.1007/978-1-4939-6952-4_14.
Microglia have increasingly been recognized as playing a wide spectrum of roles in various physiological and pathological processes in the central nervous system. Studies in the past have mostly associated individual microglial enzymes or soluble factors such as cytokines with specific functions of microglia. Stable isotope labeling with amino acids in cell culture (SILAC)-based proteomic analysis enables an unbiased, simultaneous, and global-scale analysis of the expression of thousands of proteins involved in key cellular pathways that regulate microglial activities. Primary microglia, characteristically, bear a much greater resemblance to microglia in vivo than immortalized microglial cell lines. In this chapter, we provide a detailed protocol for a de novo and uninterrupted primary culture SILAC labeling strategy (DUP-SILAC) for primary rat microglia that could be applied to the analysis of microglial involvement in various normal and disease processes.
小胶质细胞在中枢神经系统的各种生理和病理过程中所发挥的广泛作用已越来越受到认可。过去的研究大多将单个小胶质细胞酶或可溶性因子（如细胞因子）与小胶质细胞的特定功能联系起来。基于细胞培养中氨基酸的稳定同位素标记（SILAC）的蛋白质组学分析能够对参与调节小胶质细胞活性的关键细胞途径的数千种蛋白质的表达进行无偏倚、同时且全局规模的分析。与永生化小胶质细胞系相比，原代小胶质细胞在特征上与体内的小胶质细胞更为相似。在本章中，我们提供了一种用于原代大鼠小胶质细胞的从头且不间断的原代培养SILAC标记策略（DUP-SILAC）的详细方案，该方案可应用于分析小胶质细胞在各种正常和疾病过程中的参与情况。
