Improved characterization of EV preparations based on protein to lipid ratio and lipid properties.

Xabier Osteikoetxea,Andrea Balogh,Bence György,János Matkó,Tamás Géza Szabó,Barbara Sódar,Katalin Szabó-Taylor,Éva Pállinger,Ágnes Kittel,Edit Irén Buzás,Krisztina Pálóczi,Andrea Németh,Andrea Caporali

doi:10.1371/journal.pone.0121184

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.

Highlights

Extracellular vesicles (EVs) comprise a heterogeneous group of lipid bilayer enclosed vesicles released by most, if not all, cells
We propose that protein to lipid ratio, lipid bilayer order, and selected lipid composition can discriminate among differently sized EV subpopulations
In electron microphotographs some of the apoptotic bodies (APO) contained highly electron dense intravesicular content presumably corresponding to fragmented DNA

Summary

Introduction

Extracellular vesicles (EVs) comprise a heterogeneous group of lipid bilayer enclosed vesicles released by most, if not all, cells. The most extensively studied types of EVs have been classified as exosomes derived from multivesicular bodies (usually ranging from 50nm to 100nm in size [1,2,3], and microvesicles (often referred to as microparticles or ectosomes) which are directly shed from the plasma membrane (mostly with sizes of 100nm to 1μm) [1,2,4]. Cells undergoing apoptosis are known to release apoptotic vesicles (up to 5 μm, [1]). The largest apoptotic vesicles are termed apoptotic bodies [2]. Different types of EVs have been implicated with roles in intercellular communication and signaling processes such as inflammation, immune suppression, antigen presentation, tumor development, as well as in the transfer of genetic information, morphogens and signaling molecules [5]

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