Comparison of EV characterization by commercial high‐sensitivity flow cytometers and a custom single‐molecule flow cytometer

作者：James Kim, Shihan Xu, Seung‐Ryoung Jung, Alya Nguyen, Yuanhua Cheng, Mengxia Zhao, Bryant S. Fujimoto, Wyatt C. Nelson, Perry G. Schiro, Jeffrey L. Franklin, James N. Higginbotham, Robert J. Coffey, Min Yan Shi, Lucia N. Vojtech, Florian Hladik, Muneesh Tewari, John Tigges, Ionita Calin Ghiran, Tijana Jovanović‐Talisman, Louise C Laurent, Saumya Das, Olesia A. Gololobova, Kenneth Whitaker Witwer, Tuoye Xu, Al Charest, Kendall Jensen, Robert L. Raffaı̈, Jennifer Clare Jones, Joshua A Welsh, John P. Nolan, Daniel Tsun-Yee Chiu · 发表于：Journal of Extracellular Vesicles · 年份：2024 · DOI：10.1002/jev2.12498 · 被引用次数：45 · 研究领域：Extracellular vesicles in disease、Characterization and Applications of Magnetic Nanoparticles、Nanopore and Nanochannel Transport Studies

Abstract High‐sensitivity flow cytometers have been developed for multi‐parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high‐sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single‐molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di‐8‐ANEPPS and with PE‐conjugated anti‐EGFR or anti‐tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross‐calibrated, hard‐dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di‐8‐ANEPPS + /PE + particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin‐labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super‐resolution/single‐molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMF...