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.
Highlights
[Background] Small extracellular vesicles are released by virtually all mammalian cells
Most of the current methodologies used to isolate sEVs do not distinguish among different sEV subpopulation 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)
The procedure explained below uses differential ultracentrifugation followed by buoyant density flotation to allow: 1) Purification of particles associated with a lipidic membrane from non-vesicular particulate material and 2) Fractionation of distinct sEV sub-populations based on slight differences in their buoyant densities
Summary
[Background] Small extracellular vesicles (sEVs) are released by virtually all mammalian cells. Sucrose (Fisher Chemical, catalog number: S5-3) 14. 6. Add 32 ml of the collected supernatant into a single 38.5 ml ultra-clear tube. Note: This step will consume ~200 ml of conditioned medium.
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