Menck Kerstin, Bleckmann Annalen, Schulz Matthias, Ries Lena, Binder Claudia
Department of Hematology/Medical Oncology, University Medical Center Göttingen;
Department of Hematology/Medical Oncology, University Medical Center Göttingen.
J Vis Exp. 2017 Jan 6(119):55057. doi: 10.3791/55057.
The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter < 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter > 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.
细胞外囊泡（EVs）的释放是一个基本的细胞过程，发生在所有活细胞中，这些囊泡包括小的内体来源的外泌体（Exos，直径<100nm）和大的质膜来源的微囊泡（MVs，直径>100nm）。这些囊泡运输源自其母细胞的特定蛋白质、脂质和核酸，体外研究突出了它们作为细胞间通讯介质的重要性。EVs已成功地从各种体液中分离出来，尤其是血液中的EVs已被确定为癌症或传染病的有前景的生物标志物。为了能够研究血液中的MV亚群，我们提出了一种从外周血样本中标准化分离和表征MVs的方案。通过差速离心从EDTA抗凝血浆样本中沉淀出MVs，其直径通常为100 - 600nm。由于它们的尺寸较大，可以很容易地通过流式细胞术进行研究，流式细胞术是临床诊断中常用的技术，大多数实验室都有。将给出几个分离的MVs质量控制测定的例子，并介绍可用于区分血液中不同MV亚群的标志物。
