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Extracellular vesicles (EVs) are membranous vesicles that carry tremendous potential as biomarkers and therapeutic targets. Secreted by many cell types, these nanosized particles package diverse biological cargo and play a crucial part in mediating intercellular communication. However, because these intriguing messengers are so small and heterogeneous, they are also difficult to characterize.
Flow cytometry is a common technique for EV analysis1 and is widely used for EV characterization,2,3,4 but there have been several challenges associated with this method. Characterization of EVs using flow cytometry has been challenging owing to their size (30–1,000 nm),1 which is below the detection limit of regular flow cytometers (10–100 µm), and other factors such as the composition of EVs and differences in their concentrations. But innovation and standardization in flow cytometry holds promise for improved methods for their measurement.
BD Biosciences offers instrumentation and resources to tackle some of these challenges. EV researchers have utilized these new advances in innovative ways to resolve several issues that could not be previously addressed.
Read the blog entitled, “Using the latest advances in flow cytometry to tackle challenges in extracellular vesicle characterization”.
Watch the webinar organized by BD Biosciences in collaboration with Nature on the latest developments in extracellular vesicle analysis using flow cytometry with EV researchers Dr. Jonni Moore of Penn Cytomics and Cell Sorting Resource Laboratory, Dr. Terry K. Morgan of Oregon Health & Science University and Dr. Edwin van der Pol of Amsterdam University Medical Centers.
In this webinar, Dr. Edwin van der Pol demonstrates how he used flow cytometry to measure concentrations of EVs that were <100 nm in a reproducible and scalable way, while Dr. Moore and Dr. Morgan discuss mining of EV information from liquid biopsy samples and the detection of gestational phenotype differences using cell-specific EVs, respectively.
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Step 1: Prepare your instrument using standard small particle beads
Step 2: Perform start-up procedure to reduce background noise
Step 3: Apply proper controls, calibration and thresholding strategy
| 3.1 Buffy only* | 3.5 Single-stained controls* |
| 3.2 Buffer with reagents* | 3.6 Procedural controls* |
| 3.3 Unstained controls* | 3.7 Serial dilution* |
| 3.4 Isotype controls | 3.8 Detergent-treated EV samples |
STEP 4: Optional for EV size calibration
Step 5: Take advantage of the analysis power of BD FACSDiva™ and FlowJo™ Software
Output files from recommended calibration software compatible with BD FACSDiva™ and FlowJo™ Software for further analysis
Resolution of 90-nm polystyrene particles with the BD® Small Particle Detector option
Detection of 90nm polystyrene beads
Separation of 90nm polystyrene beads from noise
Side scatter sensitivity of BD® Small Particle Detector
Characterization of extracellular vesicles from human MCF7 cell line*
The BD FACSymphony™ A1 Cell Analyzer with BD® Small Particle Detector provides a dedicated high-sensitivity detector for a side scatter channel (SP SSC) designed to resolve scatter of small particles such as EVs, viral particle, exosomes and others. This offers the ability to detect particles as small as 90-nm polystyrene beads without changing the instrument setup.
FlowJo™ Software with the Rosetta Calibration plugin, along with the Rosetta Calibration product from Exometry enables calibrated size measurements of small spherical particles (<10 µm).
The plugin allows you to utilize flow cytometry standard (FCS) data captured from Rosetta Calibration beads to derive calibrated size measurements of small particles.* The Rosetta Calibration plugin provides integration between FlowJo™ 10 Software and Rosetta Calibration software.
BD flow cytometers are Class 1 Laser Products.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
