Abstract
Extracellular vesicles (EVs) are sub-micron-sized membranous spheres secreted by cells. EVs play a functional role as intercellular communicators and are associated with a number of diseases. Research into EVs is an area of growing interest due their many potential uses as therapeutic agents, as diagnostic and theranostic biomarkers, and as regulators of cellular biology. Flow cytometry is a popular method for enumerating and phenotyping EVs, even though the majority of EVs are below the detection sensitivity of most commercially available flow cytometers. Here, we present optimized protocols for EV labeling that increase the signal-to-noise ratio of EVs by removing residual antibody. Protocols for alignment of high-resolution jet-in-air flow cytometers are also provided. Published 2020. U.S. Government. Basic Protocol 1: Bulk EV staining with CFSE protein binding dye Basic Protocol 2: Antigen-specific staining of EV markers with fluorochrome-conjugated antibodies Basic Protocol 3: Astrios EQ instrument setup and sample acquisition Basic Protocol 4: Counting particles and EVs on Astrios EQ with spike-in reference beads.