Mehdiani Arash, Maier Anatol, Pinto Antonio, Barth Mareike, Akhyari Payam, Lichtenberg Artur
Research Group Experimental Surgery, Department of Cardiovascular Surgery, Medical Faculty, Heinrich-Heine-University Dusseldorf.
Research Group Experimental Surgery, Department of Cardiovascular Surgery, Medical Faculty, Heinrich-Heine-University Dusseldorf;
J Vis Exp. 2015 Jan 17(95):50974. doi: 10.3791/50974.
Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology. Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).
尽管外泌体的生物学重要性最近已获得越来越多的科学和临床关注,但关于它们复杂的途径、生物利用度以及它们在健康和疾病中的多种功能,仍有许多未知之处。当前的研究工作集中在外泌体在不同人类疾病背景下(体外以及体内)的存在和行为,尤其是在肿瘤学、妇科和心脏病学领域。不幸的是,对于外泌体分离的金标准既没有达成共识,也没有关于其定量分析方法的一致意见。由于存在许多外泌体纯化方法以及它们定量和定性分析的多种可能性,很难确定理想方法的方法组合。在此,我们展示了纳米颗粒跟踪分析(NTA),这是一种通过超速离心从人血浆中分离后用于外泌体表征的半自动方法。呈现的结果表明,这种分离方法以及外泌体平均数量和大小的测定,能提供可重复且有效的数据,其他方法如扫描电子显微镜(SEM)也证实了这一点。