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Extracellular vesicles (EVs) and other biological nanoparticles play important roles in intercellular communications and serve as potential biomarkers and therapeutics for various pathological conditions, including cancer, autoimmunity, and neurodegenerative disorders. EVs are heterogeneous, which limits the utility of analysis using bulk biochemical assays such as western blot, ELISA, and mass spectrometry. These assays report only on the total amount of EV cargo and lack valuable information about individual EVs.
Flow cytometry offers an attractive, high-throughput approach to analyzing single EVs, but their small size and dim signals pose challenges to obtaining reliable data. Recent advancements in instrument sensitivity and development of complementary, standardized assays have enabled rigorous and reproducible EV analysis by flow cytometry.
In this webinar, we will discuss how to set up the Cytek Aurora™ spectral flow cytometer with Enhanced Small Particle (ESP™) Detection Option and describe the workflow to calibrate and characterize the sensitivity and dynamic range of the instrument using Cellarcus vCal™ standards. We will also review current guidelines for measuring EVs and demonstrate how the Cellarcus Vesicle Flow Cytometry (vFC™) assay provides a practical way to comply with those guidelines to make rigorous and reproducible measurements of EV concentration, EV size (to ~50 nm diameter), and surface markers (to ~10 molecules using PE-labeled antibodies). Examples of applying that same rigor to implement multicolor analysis of EVs will be presented. In addition, we will show how fluorescence-based detection and sizing allows light scatter to be used to differentiate EV subtypes.
Key topics discussed in this webinar include:
- Harnessing spectral flow cytometry to enhance EV characterization
- Setting up and calibrating a Cytek Aurora system using Cellarcus vCal standards to characterize instrument performance and limits of detection
- Measuring and properly reporting EV size, concentration, and surface cargo using the vFC assay and suitably validated antibodies
- Researchers in the EV field
- Researchers and core facility managers interested in EVs and want to learn more about EV analysis
- Anyone interested in flow cytometry applications
1:07:35
All cells release extracellular vesicles (EVs) that can carry molecular cargo to other cells to affect their function. EVs are promising as potential biomarkers, diagnostics and therapeutics, however understanding EV origins, compositional diversity, and biological effects is limited by available methods. EVs are heterogeneous, small and difficult to measure. Conventional biochemical and molecular methods (Western blot, ELISA, PCR) that report the total signal from target in a sample cannot resolve the identity of specific EVs, while single particle approaches (NTA, RPS, AFM) lack specificity and the ability to measure EV molecular cargo. Flow cytometry is attractive as a single vesicle measurement platform, but conventional instruments and assays lack the sensitivity and specificity to resolve EVs from other small particles and background, leading to artifacts and irreproducible, uninterpretable results.
A new generation of flow cytometers with high sensitivity photodetectors, together with the use of appropriate calibration protocols, has enabled the development of assays with the sensitivity and specificity to quantitatively and reproducibly measure individual EVs. Vesicle Flow Cytometry (vFC™, Cellarcus Biosciences), uses a membrane-selective dye to detect and size individual EVs together with optimized and validated antibodies and staining protocols to measure EV cargo. In this webinar, we will cover:
- Review the aims and approaches to EV analysis, including challenges for single EV measurement
- Demonstrate vFC™ using the Beckman Coulter CytoFLEX to count, size, and measure EV cargo
- Present data on the diversity of EV surface cargo expression, including tetraspanins, integrins, and tumor-associated markers, on individual EVs, and discuss the implications for understanding EV biology