Balbi Carolina, Bolis Sara, Vassalli Giuseppe, Barile Lucio
Cellular and Molecular Cardiology Laboratory, Cardiocentro Ticino Foundation, Switzerland.
Cellular and Molecular Cardiology Laboratory, Cardiocentro Ticino Foundation, Switzerland; Molecular Cardiology Institute, Dept. of Cardiology, University of Zürich; Faculty of Biomedical Science, Università Svizzera Italiana, Switzerland;
J Vis Exp. 2019 Feb 12(144). doi: 10.3791/59128.
Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.
流式细胞术（FC）是用于细胞表面抗原标志物半定量测量的首选方法。最近，该技术已用于外周血和其他体液中包括外泌体（Exo）在内的细胞外囊泡（EV）的表型分析。EV的小尺寸要求使用检测阈值在50 - 100 nm左右的专用仪器。或者，EV可以与可通过FC检测的乳胶微珠结合。与识别EV相关标志物/分化簇CD63、CD9和CD81的抗体偶联的微珠可用于EV捕获。从CM中分离的Exo可以在经过或不经过超速离心预富集的情况下进行分析。这种方法适用于使用传统FC仪器进行的EV分析。我们的结果表明平均荧光强度（MFI）值与EV浓度之间存在线性相关性。通过超声处理破坏EV会显著降低MFI，表明该方法不会检测到膜碎片。我们报告了一种用于分析EV表面抗原的准确可靠方法，该方法可以在任何实验室轻松实施。
