You may also like
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
18:15
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.