Pedersen Ketil W, Kierulf Bente, Neurauter Axl
Thermo Fisher Scientific, Ullernchausseen 52, PO Box 114, Smestad, 0379, Oslo, Norway.
Methods Mol Biol. 2017;1660:65-87. doi: 10.1007/978-1-4939-7253-1_7.
This chapter covers magnetic bead-based isolation and analysis of the smallest members of extracellular vesicles (EVs), the exosomes (30-150 nm), generally regarded to originate from the multivesicular bodies (MVBs). Also included, are descriptions of how to prepare samples prior to isolations. The magnetic bead-based isolation workflow is dramatically shortened both by omitting the pre-enrichment step and providing an option for a very short capture time. Three direct exosome isolation strategies are described: (1) "Specific and Direct," (2) "Semi Generic and Direct" and (3) "Generic and Direct" as well as exosome release from the magnetic beads. Detailed description of downstream exosome analysis is included covering flow cytometry, Western blot and electron microscopy. Finally, a description of exosome isolation from more complex starting material including urine and serum/plasma is discussed.
本章介绍了基于磁珠的细胞外囊泡(EVs)最小成员——外泌体(30 - 150纳米)的分离和分析方法,外泌体通常被认为起源于多泡体(MVBs)。此外,还包括了分离前样品制备方法的描述。基于磁珠的分离流程通过省略预富集步骤并提供极短捕获时间的选项,显著缩短了时间。文中描述了三种直接外泌体分离策略:(1)“特异性直接法”,(2)“半通用直接法”和(3)“通用直接法”以及外泌体从磁珠上的释放。还包括了外泌体下游分析的详细描述,涵盖流式细胞术、蛋白质免疫印迹和电子显微镜检查。最后,讨论了从包括尿液和血清/血浆在内的更复杂起始材料中分离外泌体的方法。