Extracellular Vesicle Flow Cytometry

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On Demand Webinar-Flow Cytometry of Extracellular Vesicles


Flow cytometry is a powerful tool that allows for detection and characterization of multiple protein markers at single-cell resolution, both intracellularly and on the surface of many cell types. This technology can also be applied to extracellular vesicles (EVs), providing valuable information about vesicle phenotype and insights into probable function. EVs are secreted from every known cell type and facilitate the transfer of information between cells. EVs are under intensive investigation as a source of disease biomarkers, particularly in cancer and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Additionally, engineered EVs are being explored as potential therapeutic agents for regenerative medicine, vaccine adjuvants, and therapeutic drug delivery.

Explore our Poster on Exosomes by Cell Type and Process

Benefits of Flow Cytometry for Extracellular Vesicle Analysis

Illustration comparing size of cells and EVs using flow cytometry

Single Vesicle Resolution: Other methods for EV analysis (ELISA, western blot) can only provide information about the general EV population. The single-vesicle level analysis provided by flow cytometry allows determination of the heterogeneity within the overall EV population.

Multiplex Analysis: The multiplex nature of flow cytometry allows researchers to determine co-expression of different molecules on single EVs. This provides more information about populations of EVs. Since EVs are generally time consuming to isolate and analyze, it is beneficial to be able to get more information from each sample.

Bio-Techne will help streamline your EV experiments with ready-to-use kits for EV characterization by flow cytometry.

EV Source

Catalog Number

Plasma NBP3-18360
Urine NBP3-18361
Serum NBP3-18362
Cell Culture Media NBP3-18363

Following the MIFlowCyt-EV Framework for Extracellular Vesicle Flow Cytometry

In order to improve the quality of EV Flow cytometry experiments and standardize reporting, the International Society for Advancement of Cytometry (ISAC), International Society for Extracellular Vesicles (ISEV), and the International Society on Thrombosis and Haemostasis (ISTH) collaborated to develop framework published as MIFlowCyt-EV.

Bio-Techne is here to help you simplify your EV Flow Cytometry experiments with products and resources to help you achieve your research goals.

EV Isolation | Sample Preparation | Assay Controls | Instrument Considerations

Pre-analytical Variables: Extracellular Vesicle Isolation

As with any EV experiment, one of the most important considerations is the method of isolation and degree of contamination. It is recommended to continue to follow guidelines set forth by the MISEV 2018 working group.

Size-exclusion chromatography columns (SEC) can be used to isolate extracellular vesicles from biological fluids and cell culture media according to MISEV Guidelines. Bio-Techne has SEC columns and ultrafiltration devices to concentrate dilute matrices like urine and cell culture medium.


Bio-Techne Products

EV Isolation TFF-EV Concentrator
SEC columns

Find More Products for Exosome Isolation and Detection

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Sample Preparation for EV Flow Cytometry

Antibody quality is an important factor in achieving reproducible results. Bio-Techne follows the 5 Pillars of Antibody Validation established by the International Working Group for Antibody Validation (IWGAV). Common markers for EV analysis include the tetraspanins CD9, CD81, and CD63.



Sample Preparation Details Staining

Explore Knockout (KO) Validated Antibodies for Flow Cytometry

Due to their small size, fluorescence intensity from an EV will be dimmer than fluorescence intensity on a cell, so it is recommended to use antibodies conjugated to bright fluorochromes like PE, PE/Atto594, AlexaFluor® 647, and APC.

Pseudocolor flow cytometry plot showing anti-CD81 staining on human peripheral blood.

CD81 is a tetraspanin commonly found on the surface of small extracellular vesicles (sEVs). Pseudocolor flow cytometry plot showing human peripheral blood stained with APC Conjugated Human CD81 Antibody (M38) (Catalog # NBP1-44861APC). NBP1-44861 has been validated by Genetic Strategies.

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Experimental Assay Controls for EV Flow Cytometry

The MIFlowCyt-EV recommends additional controls for EV flow cytometry to ensure an accurate representation of data (listed in table below).

These controls are in addition to traditional single color and isotype controls included in every flow cytometry experiment. It is also recommended that all reagents should be filtered including antibodies, staining buffers, and sheath fluid, to better discriminate EVs from debris and protein aggregates.



Experimental Controls Buffer only
Buffer with reagents
Unstained EVs
Detergent Treated EV samples
Serial Dilutions

To build confidence in experimental results, Bio-Techne offers lyophilized and fluorescently labeled exosome standards from various cell lines and biofluids to serve as a positive control in EV flow cytometry assays. These EV standards have been isolated using ultracentrifugation, following MISEV Guidelines.


Cell Line


Healthy BioFluid Human Serum Lyophilized Fluorescent
Human Urine Lyophilized Fluorescent
Human Plasma Lyophilized Fluorescent
Healthy Human Cell Line (Embryonic Kidney Cells) HEK293 Lyophilized -
Human Glioma (Glioblastoma) U87 MG Lyophilized Fluorescent
Human Neuroblastoma SK-N-SH Lyophilized Fluorescent
Human Colon Cancer COLO1 Lyophilized Fluorescent
HCT116 Lyophilized Fluorescent
Human Prostate Cancer PC3 Lyophilized Fluorescent
LnCAP Lyophilized -

Human Lung Cancer

A549 Lyophilized Fluorescent
NCI-H1975 Lyophilized -
Human Leukemia (CML) K-562 Lyophilized Fluorescent
Human Colorectal Cancer HT29 Lyophilized -
Mouse Melanoma B16F10 Lyophilized Fluorescent

Example staining for phenotyping of extracellular vesicles by flow cytometry using exosome standards from human serum.

Histograms show expression of CD63 and CD9 on the surface of Exosome Standards (Human Serum) (Catalog #NBP2-49827) by flow cytometry. Reconstituted Exosomes can be used for profiling biomarkers. Recommended quantity: 5 μg of reconstituted Exosomes Standards for each test.

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Instrument Considerations for EV Flow Cytometry

According to MIFlowCyt-EV, Instrument calibration is a critical step to conducting a successful and reproducible EV Flow Cytometry experiment. In addition to traditional compensation and calibration bead kits, Bio-Techne offers specific support products to help complete the MIFlowCyt-EV checklist.



Bio-Techne Products

Instrument Controls Trigger Channel threshold Max Rainbow Fluorescent Particle Set (0.1-0.3 μm)
Flow rate/volumetric quantification Absolute Rainbow Cell Count Particle Set
Fluorescence calibration Ultra Rainbow Calibration ERF Particle Set

Graph showing linearity of ERF Particle set for standardization of flow cytometry units.

The Ultra Rainbow Calibration ERF Particle Set (Catalog #NBP3-11817) contains beads with 6 intensities of National Institutes of Standards and Technologies (NIST) assigned Equivalent Reference Fluorochrome (ERF) values based on a published procedure using NIST SRM 1934 and a calibrated laser-based CCD fluorimeter. ERF beads can be used to convert Mean Fluorescence Intensity (MFI) into quantitative units for standardization of flow cytometry experiments.

Traditional flow cytometers with FSC/SSC firing from the blue (488 nm) laser are not sensitive enough to detect nanometer-sized particles by light-scatter.

  1. Cytometers that can fire SSC from the violet laser (405 nm) are more sensitive for EV-based flow.
  2. Acoustic-wave cytometers can offer enhanced resolution compared to traditional hydrodynamic cytometers.

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Select References

Inglis, H., Norris, P., Danesh, A. (2015), Techniques for the analysis of extracellular vesicles using flow cytometry. J Vis Exp, 97:52484. https://doi.org/10.3791%2F52484

Welsh, J.A., Van Der Pol, E., Arkesteijn, G.J., Bremer, M., Brisson, A., Coumans, F., Dignat-George, F., Duggan, E., Ghiran, I., Giebel, B., Görgens, A., Hendrix, A., Lacroix, R., Lannigan, J., Libregts, S.F., Lozano-Andrés, E., Morales-Kastresana, A., Robert, S., De Rond, L., Tertel, T., Tigges, J., De Wever, O., Yan, X., Nieuwland, R., Wauben, M.H., Nolan, J.P. and Jones, J.C. (2020), MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. Journal of Extracellular Vesicles, 9: 1713526. https://doi.org/10.1080/20013078.2020.1713526