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Exosome Isolation by Ultracentrifugation and Precipitation and Techniques for Downstream Analyses

机译：通过超速离心和沉淀和下游分析技术的外渗分离

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摘要

Exosomes are 50‐ to 150‐nm‐diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate andanalyze 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 thesizes 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 protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. Weuse NanoSight nanoparticle tracking analysis and flow cytometry (Cytek~R) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream~R). High‐performance liquid chromatography 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 a ～2.5‐fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins.
机译：外泌体是由所有哺乳动物细胞分泌的50-至150nm的囊泡囊泡，除了成熟的红细胞，有助于体内的不同生理和病理功能。许多方法已被用于孤立和分析外来体，导致实验不一致，并提高关于如何使用不同方法获得的结果的问题。还提出了关于囊泡的各种制剂的纯度以及囊泡的存在以及脂蛋白存在的纯度。因此，调查人员经常发现鉴定其实验需求的最佳外出隔离方案挑战。我们的实验室已经比较超速离心和商业沉淀和基于柱的外出组分分离试剂盒进行外部制剂。这里，我们使用两种最常用的方法，超速离心和沉淀，然后下游分析来介绍外出分离的方案。糯纳米型纳米粒子跟踪分析和流式细胞术（Cytek〜R）测定外渗浓度和尺寸。成像流式细胞术可用于外泌体（ImageStream〜R）上的大小外肌肉和免疫蛋白酶型表面标志物。高性能液相色谱，然后用于外外馏分的纳米流动液相色谱 - 质谱（LCMS）可用于确定脂蛋白的存在，LCMS能够提供外出制剂的蛋白质组学曲线。我们发现，与超速离心相比，沉淀方法速度快，导致每毫升浓度〜2.5倍的外来浓度。两种方法在外泌体的尺寸范围内产生细胞外囊泡，并且两种制剂包括诱导蛋白。

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  • 来源
    《Current Protocols in Cell Biology》 |2020年第1期|共27页
  • 作者

    Christina Coughlan; Kimberley D. Bruce; Olivier Burgy;

  • 作者单位

    University of Colorado Alzheimer's and Cognition Center Department of Neurology Linda Crnic Institute for Down Syndrome University of Colorado Anschutz Medical Campus Aurora Colorado;

    Division of Endocrinology Metabolism and Diabetes Universityof Colorado Anschutz Medical Campus Aurora Colorado;

    Division of Pulmonary Sciences and Critical Care Medicine Department of Medicine University of Colorado Anschutz Medical Campus. INSERM U1231 Faculty of Medicine and Pharmacy University of Bourgogne‐Franche Comté Dijon France;

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  • 正文语种 eng
  • 中图分类 细胞生物学;
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