Witas Richard, Chaput Dale, Khan Hirah, Stevens Stanley M, Kang David
Department of Molecular Medicine, Byrd Alzheimer's Institute, University of South Florida, College of Medicine, Tampa, FL, USA.
Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL, USA.
Methods Mol Biol. 2017;1598:255-267. doi: 10.1007/978-1-4939-6952-4_12.
Exosomes and microvesicles are extracellular vesicles (EVs) released by most cell types. The role of EVs as a method of intercellular communication has led to these vesicles becoming a major area of interest in a variety of scientific fields including neuroscience. Emerging evidence is now demonstrating that the biomolecular composition of EVs, especially exosomes, can play a role in the progression of disease including various neurodegenerative diseases and cancer. In addition to the miRNA profiles of EVs, these vesicles also show interesting changes in protein expression profiles under different physiological and pathological conditions. Characterization of these profiles could prove valuable for both understanding disease pathogenesis and for the discovery of new biomarkers of disease. In this chapter, we describe a protocol for isolation of exosomes and microvesicles from immortalized HT22 cells and primary cortical neurons with sufficient yield and low serum contamination required for downstream analysis and label-free relative quantitation by mass spectrometry.
外泌体和微囊泡是大多数细胞类型释放的细胞外囊泡（EVs）。EVs作为一种细胞间通讯方式的作用，已使这些囊泡成为包括神经科学在内的各种科学领域的主要研究热点。新出现的证据表明，EVs，尤其是外泌体的生物分子组成，可在包括各种神经退行性疾病和癌症在内的疾病进展中发挥作用。除了EVs的miRNA谱外，这些囊泡在不同生理和病理条件下的蛋白质表达谱也呈现出有趣的变化。这些谱的表征对于理解疾病发病机制和发现新的疾病生物标志物都可能具有重要价值。在本章中，我们描述了一种从永生化HT22细胞和原代皮质神经元中分离外泌体和微囊泡的方案，该方案具有足够的产量且血清污染低，可满足下游分析和通过质谱进行无标记相对定量的要求。
