Abstract
Small extracellular vesicles (sEVs) encompass a variety of distinct vesicles that are secreted to the extracellular space. Many methodologies currently used for EV isolation (e.g., differential ultracentrifugation concluding in a high-speed pellet, precipitation by macromolecular crowding agents or size excusion chromatography–SEC) do not fractionate distinct sEV sub-populations. Samples obtained by the aforementioned methods are usually used for characterization and physiological studies. However the fraction that contains the molecule of interest or is the carrier of a specific activity is unknown. Therefore isolating distinct sEV sub-populations is critical to understand EV function. The goal of this procedure is to purify distinct sEV sub-populations based on slight differences in their buoyant density. Moreover, this technique also allows sEVs purification from vesicle-free RNA-protein complexes co-isolating in the high-speed pellet or by the use of crowding agents. This protocol describes cultivation of mammalian cells for sEV collection, sEV sedimentation, buoyant density fractionation of sEV sub-populations and immunoblots for sEV markers. This protocol can be used to fractionate distinct sEV sub-populations produced by a variety of mammalian cells.
Keywords: Extracellular vesicles, Differential ultracentrifugation, Buoyant density, Exosomes, RNA-protein complexes, OptiPrep
Background
Small extracellular vesicles (sEVs) are released by virtually all mammalian cells. They have been proposed to play a variety of roles ranging from immunity to cancer metastasis (Pegtel et al., 2010; Mittelbrunn et al., 2011; Montecalvo et al., 2012; Fong et al., 2015; Dickman et al., 2017). Most of the current methodologies used to isolate sEVs however do not distinguish among different sEV sub-population nor do they purify sEVs from co-isolating protein and RNA-protein complexes (Temoche-Diaz et al., 2019; Kowal et al., 2016; Willms et al., 2016; Jeppesen et al., 2019). Therefore the sedimentable fraction or specific vesicle species that promotes a particular physiological response remains unknown (Shurtleff et al., 2018). A methodology that allows purification of sEVs away from contaminants and fractionation of distinct sEV sub-populations is of broad utility. The procedure explained below uses differential ultracentrifugation followed by buoyant density flotation to allow: 1) Purification of particles associated with a lipidic membrane (sEVs) from non-vesicular particulate material and 2) Fractionation of distinct sEV sub-populations based on slight differences in their buoyant densities. The purified sEV fractions are of suitable purity for use in physiological and/or characterization experiments.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
This protocol assumes the users have knowledge of how to perform immunoblots and analyze the data.
Recipes
Acknowledgments
We would like to thank current and former Schekman lab members who provided technical advice in the development of this protocol. We also acknowledge the Howard Hughes Medical Institute for funding. This protocol has been adapted from Temoche-Diaz et al., 2019.
Competing interests
The authors declare no competing interests.
References
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