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.
Keywords: Mass spectrometry; Microglia; Neuroinflammation; Pathway profiling; SILAC.