Extracellular Vesicle Flow Cytometry
Flow cytometry is a powerful tool that allows the 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 EV phenotype and function.
Since EVs are secreted from every known cell type and facilitate the transfer of information between cells, they are of great interest as a potential 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. Therefore, flow cytometry of EVs could be a useful tool to identify novel EV biomarkers for disease.
Bio-Techne can help streamline your EV experiments with ready-to-use kits for EV characterization by flow cytometry.
| EV Source | Catalog # |
|---|---|
| Plasma | NBP3-18360 |
| Urine | NBP3-18361 |
| Serum | NBP3-18362 |
| Cell Culture Media | NBP3-18363 |
Benefits of Flow Cytometry for Extracellular Vesicle Analysis
- 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 for the determination of the heterogeneity within the overall EV population.
- Multiplex Analysis: The multiplex nature of flow cytometry allows researchers to explore co-expression of different molecules on single EVs, providing more information about populations of EVs. Since EVs are generally time consuming to isolate and analyze, it is beneficial to be able to obtain more information from each sample. Use our Flow Cytometry Panel Builder to help you select the right fluorochromes for multicolor flow cytometry.
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) have collaborated to develop framework published as MIFlowCyt-EV.
Bio-Techne recognizes the need for compliance with the MIFlowCyt-EV framework and is here to help you simplify your EV Flow Cytometry experiments, with a range of products and resources to help you achieve your research goals.
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.
Products for EV Isolation
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.
Owing 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.
The MIFlowCyt-EV recommends additional controls for EV flow cytometry to ensure an accurate representation of the data (listed in table below).
These controls are in addition to traditional single color and isotype controls that are 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 include:
- 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 positive controls in EV flow cytometry assays. These EV standards have been isolated using ultracentrifugation, following MISEV Guidelines.
| EV Source | Cell Line | Format |
|---|---|---|
| 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 |
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.
Instrument Controls
| Detail | Product |
|---|---|
| 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 |
The type of flow cytometer to be used for EVs is an essential consideration, as traditional hydrodynamic flow cytometers with forward scatter (FSC) and side scatter (SSC) firing from the blue (488 nm) laser are not sensitive enough to detect nanometer-sized particles by light-scatter. Alternative types of flow cytometer that are more suited towards EV-based flows include:
- Cytometers that can fire SSC from the violet laser (405 nm), offering increased sensitivity,
- Acoustic-wave cytometers generating ultrasonic waves, offering enhanced resolution.
- Inglis, H. et al. (2015) Techniques for the analysis of extracellular vesicles using flow cytometry. J Vis Exp 97:52484. PMID: 25867010.
- Welsh, J.A. et al. (2020) MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. Journal of Extracellular Vesicles 9:1713526. PMID: 32128070.