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Isolation and Quantification of Plant Extracellular Vesicles.

Bio-protocol (2017-09-05)
Brian D Rutter, Katie L Rutter, Roger W Innes
ABSTRACT

Extracellular vesicles (EVs) play an important role in intercellular communication by transporting proteins and RNA. While plant cells secrete EVs, they have only recently been isolated and questions regarding their biogenesis, release, uptake and function remain unanswered. Here, we present a detailed protocol for isolating EVs from the apoplastic wash of Arabidopsis thaliana leaves. The isolated EVs can be quantified using a fluorometric dye to assess total membrane content.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
MES hydrate, ≥99.5% (titration)
Sigma-Aldrich
Murashige and Skoog Basal Salt Mixture (MS), powder, suitable for plant cell culture
Sigma-Aldrich
Protease Inhibitor Cocktail, for plant cell and tissue extracts, DMSO solution
Sigma-Aldrich
OptiPrep Density Gradient Medium, used for cell and subcellular organelle isolation
Sigma-Aldrich
Dipropyl disulfide, 98%
Sigma-Aldrich
Trizma® base, Primary Standard and Buffer, ≥99.9% (titration), crystalline
Sigma-Aldrich
Calcium chloride, anhydrous, granular, ≤7.0 mm, ≥93.0%