Isolation of Dendropanax morbifera Leaf Extracellular Vesicles

We collected fresh leaves of Dendropanax morbifera from Bogil Island, which is located in Wando-gun, Jeollanam-do, South Korea. Unlike the general method for isolating plant vesicles [39 (link),40 (link),41 (link)], we have developed a method for isolating EVs from Dendropanax morbifera leaf to facilitate industrial application. Dendropanax morbifera-derived EVs were isolated by processing the leaves with a mixer grinder plus extractor, passing the resulting crude leaf extract through filter paper, and centrifuging the obtained extract at 10,000× g for 10 min. Then, large debris was removed by filtering the supernatant through a 0.22-μm membrane, and then the filtered EVs were concentrated by centrifuging the sample at 5000× g for 10 min at 4 °C in an Amicon Ultra-4 PL 100 K concentrator (Merck Millipore, Darmstadt, Germany) [5 (link)]. After isolating LEVs, we measured the protein concentration using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and prepared it by dilution with distilled water to calculate equal concentration of LEVs with and without preservatives. After centrifugation, the protein concentration of EVs was estimated using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).