The Exploration and Establishment of a Method to Detect Extracellular Vesicles by Flow Cytometry
GUO Chun1, WANG Jiajia1, LI Yanwei1, XING Yueting1, SONG Xinghui1, HUANG Yingying1, YE Xiaokang2*
This study was aimed to explore the standardized process for the analysis of EVs (extracellular vesicles) and establish a highly efficient detection method for EVs, which provided technical support for the function and clinical translational research of EVs. EVs were isolated by classic ultracentrifugation. The flow cytometric detection parameters were setted up and optimized with the help of polystyrene microspheres of known diameter, and then the application of scattered light signal and fluorescence signal were combined to conduct dual-parameter analysis and identification of EVs. Finally the EVs were observed by electron microscope and WB (Western blot) to verify the identification results. The results of the flow cytometry experiment showed that the 100 nm particle signal could be clearly separated from the noise signal with reference to the conditions setted by the nanospheres. There was a good linear correlation between the gradient of the dilution of the microspheres and the detected concentration, which allowed quantitative analysis of EVs. The fluorescent staining results showed that EVs were double positive for CFSE (protein binding dye) and FM1-43 (lipophilic dye), accounting for about 3%. Its shape and diameter were verified by electron microscopy and consistent with those reported in the literature. The study explored the standardization process for flow cytometric detection method of EVs through optimized flow cytometric scheme and dual fluorescence parameter analysis and identification, and established an efficient and accurate EVs flow cytometric detection method.
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