Protocol for the isolation of human neutrophil-derived extracellular vesicles for characterization and functional experimentation.

Small extracellular vesicles (EVs) derived from neutrophils play an important role in the remodeling of extracellular matrices in human health and disease. Here, we present a protocol for isolating EVs secreted from human neutrophils either ex vivo or within circulation. We describe steps for neutrophil and plasma isolation, neutrophil activation, and EV isolation and enumeration. We then detail procedures for isolating CD66b(+) EVs using magnetic bead pull-down and subsequent characterization by flow cytometry, western blot, and neutrophil elastase activity assays. For complete information on the generation and use of this protocol, please refer to Genschmer et al.(1).