Isolation of Proteins and Extracellular Vesicles Secreted by Pollen of Actinidia chinensis L. During In Vitro Germination.

Extracellular vesicles (EVs) are membrane-bound particles secreted into the extracellular space by cells of any living kingdom. Plant-derived EVs (PDEVs) in particular are regarded as promising nutraceutical and drug carriers, but their purification is hindered by the contamination from intracellular and intramural vesicles, which is inherently associated with the existing PDEVs isolation protocols. In the last decade, pollen has been proven to release EVs, called pollensomes, during in vitro germination. When secreted in a liquid culture medium, pollensomes can be easily isolated with minimal cellular contamination, thus representing a promising source of purified PDEVs either for fundamental research studies or for biomedical applications. Here, we describe a straightforward protocol to isolate EVs smaller than 220 nm (called small EVs) from Actinidia chinensis L. germinated pollen, using sequential filtration and ultracentrifugation at 100,000× g. We also detail procedures to extract proteins secreted by pollen during germination, either via EVs or by EV-independent pathways, for further analyses of the pollen secretome.