La Trobe

File(s) under permanent embargo

A protocol for isolation and proteomic characterization of distinct extracellular vesicle subtypes by sequential centrifugal ultrafiltration

posted on 06.08.2021, 07:15 authored by Rong Xu, Richard SimpsonRichard Simpson, David GreeningDavid Greening
Scientific and clinical interest in extracellular vesicles (EVs) has increased rapidly as evidence mounts that they may constitute a new signaling paradigm. Recent studies have highlighted EVs carry preassembled complex biological information that elicit pleiotropic responses in target cells. It is well recognized that cells secrete essentially two EV subtypes that can be partially separated by differential centrifugation (DC): the larger size class (referred to as “microvesicles” or “shed microvesicles,” sMVs) is heterogeneous (100–1500 nm), while the smaller size class (referred to as “exosomes”) is relatively homogeneous in size (50–150 nm). A key issue hindering progress in understanding underlying mechanisms of EV subtype biogenesis and cargo selectivity has been the technical challenge of isolating homogeneous EV subpopulations suitable for molecular analysis. In this protocol we reveal a novel method for the isolation, purification, and characterization of distinct EV subtypes: exosomes and sMVs. This method, based on sequential centrifugal ultrafiltration (SCUF), affords unbiased isolation of EVs from conditioned medium from a human colon cancer cell model. For both EV subtypes, this protocol details extensive purification and characterization based on dynamic light scattering, cryoelectron microscopy, quantitation, immunoblotting, and comparative label-free proteome profiling. This analytical SCUF method developed is potentially scalable using tangential flow filtration and provides a solid foundation for future in-depth functional studies of EV subtypes from diverse cell types.


Publication Date


Book Title

Exosomes and Microvesicles: Methods and Protocols


Hill AF


Humana Press

Place of publication

New York, NY, USA


Methods in Molecular Biology




26p. (p. 91-116)



Rights Statement

The Author reserves all moral rights over the deposited text and must be credited if any re-use occurs. Documents deposited in OPAL are the Open Access versions of outputs published elsewhere. Changes resulting from the publishing process may therefore not be reflected in this document. The final published version may be obtained via the publisher’s DOI. Please note that additional copyright and access restrictions may apply to the published version.