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Isolation and characterization of extracellular vesicle subpopulations from tissues
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Isolation and characterization of extracellular vesicle subpopulations from tissues
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Journal Article
Isolation and characterization of extracellular vesicle subpopulations from tissues
2021
Overview
Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.
This protocol describes how to isolate up to six different subpopulations of extracellular vesicles (EVs) from tissues. The procedure includes detailed instructions for EV characterization using electron microscopy, RNA and protein analysis.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ Animals
/ Biomedical and Life Sciences
/ Cancer
/ Centrifugation, Density Gradient - methods
/ Computational Biology/Bioinformatics
/ Extracellular Vesicles - metabolism
/ Extracellular Vesicles - physiology
/ Humans
/ Identification and classification
/ Lipids
/ Melanoma
/ Methods
/ Microscopy, Electron - methods
/ Proteins
/ Proteins - isolation & purification
/ Protocol
/ Tissues
/ Triiodobenzoic Acids - chemistry
/ Ultracentrifugation - methods
/ Vesicles
