A compendium of single extracellular vesicle flow cytometry
Welsh, Joshua A; Arkesteijn, Ger J A; Bremer, Michel; Cimorelli, Michael; Dignat-George, Françoise; Giebel, Bernd; Görgens, André; Hendrix, An; Kuiper, Martine; Lacroix, Romaric; Lannigan, Joanne; van Leeuwen, Ton G; Lozano-Andrés, Estefanía; Rao, Shoaib; Robert, Stéphane; de Rond, Leonie; Tang, Vera A; Tertel, Tobias; Yan, Xiaomei; Wauben, Marca H M; Nolan, John P; Jones, Jennifer C; Nieuwland, Rienk; van der Pol, Edwin
(2023) Journal of Extracellular Vesicles, volume 12, issue 2, pp. 1 - 70
(Article)
Abstract
Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to
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detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.
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Keywords: MIFlowCyt-EV, calibration, extracellular vesicles, flow cytometry, microparticles, nanoparticles, standardization, Histology, Cell Biology
ISSN: 2001-3078
Publisher: Co-Action Publishing
Note: Funding Information: André Görgens has equity interest in and is a consultant for Evox Therapeutics Ltd, Oxford, UK. André Görgens is inventor on patents and patent applications related to extracellular vesicle engineering, manufacturing, and analysis. An Hendrix is an inventor on patents and patent applications related to extracellular vesicle products. Edwin van der Pol is co‐founder and shareholder of Exometry BV. Joshua A. Welsh and Jennifer C. Jones are inventors on NIH patents and patent applications related to extracellular vesicle assays. John P. Nolan is CEO of Cellarcus Biosciences. Joanne Lannigan is a paid consultant for EV development work for Cytek Biosciences. Romaric Lacroix and Françoise Dignat‐George declare that they have patents on extracellular vesicles assays and have received funding from the companies Stago and Beckman Coulter. Xiaomei Yan declares a competing financial interest as a cofounder and shareholder of NanoFCM Inc. Funding Information: We thank Dr. Aleksandra Gąsecka for providing the data for Figure 13. André Görgens, Joshua A. Welsh: International Society for Advancement of Cytometry (ISAC) Marylou Ingram Scholarship 2019–2023; Vera A. Tang: ISAC Shared Resource Lab Emerging Leader 2018–2023; Edwin van der Pol: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (grant numbers VENI 15924 & VIDI 19724); Joshua A. Welsh, Jennifer C. Jones: Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, Center for Cancer Research, NIH ZIA BC011502/3 and NIH UG3/UH3 TR002881; Michael Cimorelli: National Science Foundation (Award #1829436) and the Netherlands-America Foundation; Martine Kuiper, Rienk Nieuwland: This work is part of the project 18HLT01 METVES II and received funding from the EMPIR programme co-financed by the Participating States and from the European Union's Horizon 2020 research and innovation programme. Estefanía Lozano-Andrés is supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 722148. Romaric Lacroix; Stéphane Robert and Françoise Dignat-George: French Research National Agency ANR-18RHUS-0015; Xiaomei Yan: National Natural Science Foundation of China (21627811 and 21934004); Funding Information: We thank Dr. Aleksandra Gąsecka for providing the data for Figure 13 . André Görgens, Joshua A. Welsh: International Society for Advancement of Cytometry (ISAC) Marylou Ingram Scholarship 2019–2023; Vera A. Tang: ISAC Shared Resource Lab Emerging Leader 2018–2023; Edwin van der Pol: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (grant numbers VENI 15924 & VIDI 19724); Joshua A. Welsh, Jennifer C. Jones: Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, Center for Cancer Research, NIH ZIA BC011502/3 and NIH UG3/UH3 TR002881; Michael Cimorelli: National Science Foundation (Award #1829436) and the Netherlands‐America Foundation; Martine Kuiper, Rienk Nieuwland: This work is part of the project 18HLT01 METVES II and received funding from the EMPIR programme co‐financed by the Participating States and from the European Union's Horizon 2020 research and innovation programme. Estefanía Lozano‐Andrés is supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No 722148. Romaric Lacroix; Stéphane Robert and Françoise Dignat‐George: French Research National Agency ANR‐18RHUS‐0015; Xiaomei Yan: National Natural Science Foundation of China (21627811 and 21934004); Publisher Copyright: © 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.
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