Protocol for ultracentrifugation-based isolation and characterization of extracellular vesicles from FGF2-primed astrocytes

Extracellular vesicles (EVs) mediate intercellular communication by transferring bioactive molecular cargo. We present a protocol for isolating EVs from fibroblast growth factor-2 (FGF2)-primed primary mouse astrocytes using differential ultracentrifugation. We describe steps for primary astrocyte culture, FGF2 priming, EV isolation, and comprehensive characterization encompassing transmission electron microscopy (TEM). We then detail procedures for nanoparticle tracking analysis (NTA), western blotting, and mass spectrometry-based proteomic analysis. EVs isolated by this approach protect against mitochondrial and synaptic toxicity in Parkinson's disease models. For complete details on the use and execution of this protocol, please refer to Wen et al. 1