Volume 95, Issue 1 e81
PROTOCOL

Detection and Sorting of Extracellular Vesicles and Viruses Using nanoFACS

Aizea Morales-Kastresana

Aizea Morales-Kastresana

Laboratory of Pathology, Translational Nanobiology Section, Centre for Cancer Research, National Institute of Health, National Institutes of Health, Bethesda, Maryland

Joint first authors.

Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Writing - original draft, Writing - review & editing

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Joshua A. Welsh

Joshua A. Welsh

Laboratory of Pathology, Translational Nanobiology Section, Centre for Cancer Research, National Institute of Health, National Institutes of Health, Bethesda, Maryland

Joint first authors.

Contribution: Formal analysis, ​Investigation, Methodology, Software, Validation, Visualization, Writing - review & editing

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Jennifer C. Jones

Corresponding Author

Jennifer C. Jones

Laboratory of Pathology, Translational Nanobiology Section, Centre for Cancer Research, National Institute of Health, National Institutes of Health, Bethesda, Maryland

Joint first authors.

Corresponding author: [email protected]

Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, ​Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing - review & editing

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First published: 17 December 2020
Citations: 2

Abstract

Extracellular vesicles (EVs) are sub-micron-sized membranous spheres secreted by cells. EVs play a functional role as intercellular communicators and are associated with a number of diseases. Research into EVs is an area of growing interest due their many potential uses as therapeutic agents, as diagnostic and theranostic biomarkers, and as regulators of cellular biology. Flow cytometry is a popular method for enumerating and phenotyping EVs, even though the majority of EVs are below the detection sensitivity of most commercially available flow cytometers. Here, we present optimized protocols for EV labeling that increase the signal-to-noise ratio of EVs by removing residual antibody. Protocols for alignment of high-resolution jet-in-air flow cytometers are also provided. Published 2020. U.S. Government.

Basic Protocol 1: Bulk EV staining with CFSE protein binding dye

Basic Protocol 2: Antigen-specific staining of EV markers with fluorochrome-conjugated antibodies

Basic Protocol 3: Astrios EQ instrument setup and sample acquisition

Basic Protocol 4: Counting particles and EVs on Astrios EQ with spike-in reference beads