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Related Experiment Video

Updated: Nov 25, 2025

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry
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Detection and Sorting of Extracellular Vesicles and Viruses Using nanoFACS.

Aizea Morales-Kastresana1, Joshua A Welsh1, Jennifer C Jones1

  • 1Laboratory of Pathology, Translational Nanobiology Section, Centre for Cancer Research, National Institute of Health, National Institutes of Health, Bethesda, Maryland.

Current Protocols in Cytometry
|December 17, 2020
PubMed
Summary

Optimized protocols enhance extracellular vesicle (EV) detection using flow cytometry. These methods improve signal-to-noise ratios for better EV characterization and analysis.

Keywords:
EV sortingEVsextracellular vesiclesflow cytometryflow virometrynano flow cytometrynanoFACSvirus sorting

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Immunology

Background:

  • Extracellular vesicles (EVs) are crucial intercellular communicators implicated in various diseases.
  • EV research is expanding due to their therapeutic and diagnostic potential.
  • Flow cytometry is widely used for EV analysis, but faces limitations with sub-micron particle detection.

Purpose of the Study:

  • To present optimized protocols for extracellular vesicle (EV) labeling and flow cytometer setup.
  • To enhance the signal-to-noise ratio for improved EV detection and phenotyping.
  • To provide standardized methods for high-resolution flow cytometric analysis of EVs.

Main Methods:

  • Optimized protocols for bulk EV staining using CFSE dye.
  • Antigen-specific staining of EV markers with fluorochrome-conjugated antibodies.
  • High-resolution jet-in-air flow cytometer (Astrios EQ) setup and sample acquisition protocols.
  • Methods for particle counting using spike-in reference beads.

Main Results:

  • Protocols effectively increase the signal-to-noise ratio of extracellular vesicles (EVs).
  • Optimized labeling and antibody removal enhance EV detectability.
  • Standardized instrument setup ensures reliable and reproducible EV quantification.

Conclusions:

  • The presented protocols significantly improve the flow cytometric analysis of extracellular vesicles (EVs).
  • These methods facilitate more accurate enumeration and phenotyping of EVs, advancing their study.
  • This work supports the use of flow cytometry for robust EV biomarker discovery and characterization.