Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties

Abstract

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.

MYC Mediates Large Oncosome-Induced Fibroblast Reprogramming in Prostate Cancer

“…Tunable resistive pulse sensing measurements EV preparations were submitted to tunable resistive pulse sensing (TRPS) analysis using a qNano instrument (IZON Science) as described previously (15).…”

Differential detergent sensitivity of extracellular vesicle subpopulations

“…5 Briefly, EXOs were found to be more liquid ordered while both APOs and MVs had similar and lower membrane liquid orders. 5 This is in accordance with the findings that liquid ordered as opposed to liquid-disordered membranes are resistant to detergents. 18,19 Finally, our data may have impact on EV research beyond TRPS and flow cytometry.…”

“…5,13 At least 500 particles were counted (except when lysis was observed in which case one minute recording time was used regardless of particle counts). NP100, NP400, NP800 and NP2000 nanopore membranes stretched between 43-47 mm were used.…”

Thematic Review Series: Exosomes and Microvesicles: Lipids as Key Components of their Biogenesis and Functions, Cholesterol and the journey of extracellular vesicles

“…Indirect evidence for the elevated cholesterol content comes from the observation that EVs from cell line supernatant (15) and from human body fluids (16,17) contain cholesterol-binding by guest, on April 27, 2019 www.jlr.org Downloaded from 6 proteins such as prominin 1 (CD133). Membranes of EVs may also contain cholesterol-rich lipid rafts: EVs were bound by an antibody against clustered cholesterol (18) and they contained detergent-resistant membrane fractions featuring the raft marker stomatin and enhanced cholesterol levels (19). Treatment of cells with ostreolysin A, a fungal toxin that binds to sphingomyelin/cholesterol-rich membrane domains, induced membrane shedding and the generation of EVs with a two-fold higher cholesterol content than the cell lysate (20).…”