Density-Based Fractionation of Cell-Conditioned Medium to Prepare Proteomics Grade Extracellular Vesicles

Quentin Roux; Sarah Deville; An Hendrix

doi:10.1007/978-1-0716-3457-8_14

Density-Based Fractionation of Cell-Conditioned Medium to Prepare Proteomics Grade Extracellular Vesicles

Methods Mol Biol. 2023:2718:253-269. doi: 10.1007/978-1-0716-3457-8_14.

Authors

Quentin Roux^{1

2}, Sarah Deville^{1

2}, An Hendrix^{3

4}

Affiliations

¹ Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.
² Cancer Research Institute Ghent, Ghent, Belgium.
³ Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium. an.hendrix@ugent.be.
⁴ Cancer Research Institute Ghent, Ghent, Belgium. an.hendrix@ugent.be.

PMID: 37665464
DOI: 10.1007/978-1-0716-3457-8_14

Abstract

The identification of the molecular composition of extracellular vesicles (EV) by omics approaches, including proteomics, requires the separation of EV from non-EV confounding factors present in the source biofluid. In this protocol, we present the sequential implementation of density gradient ultracentrifugation and size-exclusion chromatography to prepare EV from cell-conditioned medium with high specificity and repeatability. This approach enables the recovery of intact purified EV suited for downstream functional assays and biomarker discovery by omics approaches.

Keywords: Cell culture; Density gradient; Extracellular vesicles; Proteomics; Size-exclusion chromatography.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Fractionation
Centrifugation, Density Gradient
Chromatography, Gel
Culture Media, Conditioned
Cytological Techniques* / methods
Extracellular Vesicles* / chemistry
Humans
Proteomics

Substances

Culture Media, Conditioned