Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses.
Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses.
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DOI：
10.1002/cpcb.110
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发表时间：
2020-09
影响因子：
--
通讯作者：
Potter H
中科院分区：
文献类型：
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作者：
Coughlan C;Bruce KD;Burgy O;Boyd TD;Michel CR;Garcia-Perez JE;Adame V;Anton P;Bettcher BM;Chial HJ;Königshoff M;Hsieh EWY;Graner M;Potter H
外泌体是由除成熟红细胞外的所有哺乳动物细胞分泌的直径为50-150 nm的细胞外囊泡，并且有助于体内的多种生理和病理功能。许多方法已被用于分离和分析外泌体，导致实验之间的不一致，并提出了如何比较使用不同方法获得的结果的问题。关于各种制剂的纯度、囊泡的大小和类型以及脂蛋白的存在也提出了问题。因此，研究人员经常发现确定最佳外泌体分离方案以满足其实验需求是具有挑战性的。我们的实验室已经比较了用于外泌体制备的超离心和商业沉淀以及基于柱的外泌体分离试剂盒。在这里，我们介绍了我们对使用两种最常用的方法分离的外泌体的研究，超离心和沉淀，然后进行下游分析。我们使用NanoSight纳米颗粒跟踪分析（NTA）和流式细胞术（Cytek®）来确定外泌体浓度和大小。成像流式细胞术可用于测定外来体上的表面标志物的大小和免疫表型（ImageStream）。可以使用外泌体级分的高效液相色谱法（HPLC）和随后的纳米流液相色谱质谱法（LCMS）来确定脂蛋白的存在，其中LCMS能够提供外泌体制剂的蛋白质组学谱。我们发现沉淀方法比超离心法快六倍，并且导致每毫升外泌体浓度高约2.5倍。两种方法都产生了在外来体大小范围内的EV，并且两种制剂都包含脱辅基蛋白。
Exosomes are 50-150 nm diameter extracellular vesicles secreted by all mammalian cell, except mature red blood cells, and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles, and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation and column-based exosome isolation kits for exosome preparation. Here, we present our study of exosomes isolated using two of the most commonly-used methods, ultracentrifugation and precipitation, followed by downstream analyses.. We used NanoSight nanoparticle tracking analysis (NTA) and Flow cytometry (Cytek® ) to determine exosome concentrations and size. Imaging flow cytometry can be utilized to both size and immunophenotype surface markers on exosomes (ImageStream). High-performance liquid chromatography (HPLC) followed by nano-flow liquid chromatography mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster, and resulted in ~2.5 fold higher concentration of exosomes per ml compared to ultracentrifugation. Both methods yielded EVs in the size range of exosomes and both preparations included apoproteins.