Protocol for isolating plant-derived extracellular vesicles

Plant-derived extracellular vesicles (PEVs) are nanosized, lipid bilayer-enclosed vesicles secreted by plant cells into the extracellular space, playing critical roles in intercellular communication. Here, we present a comprehensive and reproducible protocol for isolating PEVs from diverse plant species. Our integrated approach combines differential centrifugation, ultrafiltration, ultracentrifugation, and sucrose density gradient centrifugation to achieve efficient enrichment and purification of PEVs while effectively removing cellular debris. Finally, we characterize the isolated PEVs in terms of morphology, particle size distribution, and zeta potential.