In this tutorial, Dr. John Nolan will describe fluorescence based EV size measurement. In this talk we will describe vFC™, an approach to characterizing EVs against the background of the current MISEV guidelines. We will go through a typical workflow including characterizing your instrument, running a vesicle size standard, and calibrating fluorescence measurements into surface area (and diameter). We will wrap up with a comparison to other approaches and a few recommendations on integrating methods for rigorous, reproducible measurements.
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
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