Protocol for the separation of extracellular vesicles by ultracentrifugation from in vitro cell culture models

Extracellular vesicles (EVs) play key roles in transporting key molecular constitu-ents as cargo for extracellular trafficking. While several approaches have been developed to extract EVs from mammalian cells, the specific method of EV isola-tion can have a profound effect on membrane integrity and yield. Here, we describe a step-by-step procedure to separate EVs from adherent epithelial cells using differential ultracentrifugation. Separated EVs can be further analyzed by immunoblotting, mass spectrometry, and transmission electron microscopy to derive EV yield and morphology. For complete details on the use and execution of this protocol, please refer to Brown et al. (2019).