Coelho Carolina, Vij Raghav, Smith Daniel Q, Brady Nathan R, Hamacher-Brady Anne, Casadevall Arturo
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore MD, USA.
Bio Protoc. 2020 Jan 20;10(2):e3502. doi: 10.21769/BioProtoc.3502.
Extracellular vesicles (EVs) are produced by all domains of life including Bacteria, Archaea and Eukarya. EVs are critical for cellular physiology and contain varied cargo: virulence factors, cell wall remodeling enzymes, extracellular matrix components and even nucleic acids and metabolites. While various protocols for isolating EVs have been established for mammalian cells, the field is actively developing tools to study EVs in other organisms. In this protocol we describe our methods to perform density gradient purification of EVs in bacterial cells, allowing for separation of EV subpopulations, followed by protection assays for EV cargo characterization. Furthermore, we devised a protocol which incorporates a fluorescent conjugate of fatty acids into EVs, the first to allow -cell EV tracking to observe release of EVs, including during infection of mammalian cells by pathogenic bacteria. These protocols are powerful tools for EV researchers as they enable the observation of EV release and the study of the mechanisms of their formation and release.
细胞外囊泡（EVs）由包括细菌、古菌和真核生物在内的所有生命域产生。EVs对细胞生理至关重要，并且包含各种各样的货物：毒力因子、细胞壁重塑酶、细胞外基质成分，甚至核酸和代谢物。虽然已经为哺乳动物细胞建立了各种分离EVs的方案，但该领域正在积极开发用于研究其他生物体中EVs的工具。在本方案中，我们描述了在细菌细胞中对EVs进行密度梯度纯化的方法，以便分离EV亚群，随后进行用于EV货物表征的保护测定。此外，我们设计了一种方案，该方案将脂肪酸的荧光共轭物掺入EVs中，这是第一个允许对细胞EV进行追踪以观察EVs释放的方案，包括在病原菌感染哺乳动物细胞期间。这些方案对于EV研究人员来说是强大的工具，因为它们能够观察EV释放并研究其形成和释放机制。
