Isolation of Exosome-Like Vesicles from Plants by Ultracentrifugation on Sucrose/Deuterium Oxide (D2O) Density Cushions
Exosomes are nanovesicles of endocytic origin that are about 30–100 nm in diameter, surrounded by a lipid bilayer membrane, and contain proteins, nucleic acids, and other molecules. Mammalian cells- and biological fluids-derived exosomes have become the subject for a wide range of investigations in biological and biomedical sciences. More recently, a new interest is on the verge of rising: the presence of nanovesicles in plants. Lipoprotein vesicles from apoplastic fluid and exosome-like vesicles (ELVs) from fruit juice have been isolated and shown that they could be loaded with drugs and uptaken by recipient cells. In order to explore and analyze the contents and functions of ELVs, they must be isolated and purified with intense care. Isolation of ELVs can be a tedious process and often characterized by the co-purification of undesired contaminants. Here we describe a method which isolates ELVs based on their buoyant density. The method utilizes differential centrifugation in step 1 and 1 and 2 M sucrose/deuterium oxide double-cushion ultracentrifugation in step 2, to purify two diverse ELV subpopulations. In this method fruit juice is used as an example of starting material, although this protocol can be used for the isolation of vesicles from apoplastic fluid too. The quality and the quantity of ELV preparations have been found appropriate for downstream biological and structural studies, like proteomics, transcriptomics, and lipidomics.
Key wordsExtracellular vesicles Exosome-like vesicles Apoplastic vesicles Plant tissue Juice Purification Isolation Ultracentrifugation Differential centrifugation Sucrose gradient centrifugation
- 1.Yáñez-Mó M, Siljander PR-M, Andreu Z, Zavec AB, Borràs FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J, Colás E, Cordeiro-da Silva A, Fais S, Falcon-Perez JM, Ghobrial IM, Giebel B, Gimona M, Graner M, Gursel I, Gursel M, Heegaard NHH, Hendrix A, Kierulf P, Kokubun K, Kosanovic M, Kralj-Iglic V, Krämer-Albers E-M, Laitinen S, Lässer C, Lener T, Ligeti E, Linē A, Lipps G, Llorente A, Lötvall J, Manček-Keber M, Marcilla A, Mittelbrunn M, Nazarenko I, Nolte-‘t Hoen ENM, Nyman TA, Driscoll L, Olivan M, Oliveira C, Pállinger É, del Portillo HA, Reventós J, Rigau M, Rohde E, Sammar M, Sánchez-Madrid F, Santarém N, Schallmoser K, Stampe Ostenfeld M, Stoorvogel W, Stukelj R, Van der Grein SG, Vasconcelos MH, Wauben MHM, De Wever O (2015) Biological properties of extracellular vesicles and their physiological functions. Journal of Extracellular Vesicles 4. doi: 10.3402/jev.v4.27066
- 7.Ju S, Mu J, Dokland T, Zhuang X, Wang Q, Jiang H, Xiang X, Deng ZB, Wang B, Zhang L, Roth M, Welti R, Mobley J, Jun Y, Miller D, Zhang HG (2013) Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis. Mol Ther 21(7):1345–1357CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Joosten MAJ (2012) Isolation of apoplastic fluid from leaf tissue by the vacuum infiltration-centrifugation technique. In: Bolton MD, Thomma BPHJ (eds) Plant fungal pathogens, vol 835, Methods in molecular biology. Humana, New York, NY, pp 603–610. doi: 10.1007/978-1-61779-501-5_38 CrossRefGoogle Scholar