title: 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.
authors: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Xabier
authors: X
authors: Osteikoetxea
authors: Department of Immunology, Eötvös Loránd University, Budapest, Hungary.
authors: Andrea
authors: A
authors: Balogh
authors: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Katalin
authors: K
authors: Szabó-Taylor
authors: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Andrea
authors: A
authors: Németh
authors: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Tamás Géza
authors: TG
authors: Szabó
authors: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Krisztina
authors: K
authors: Pálóczi
authors: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Barbara
authors: B
authors: Sódar
authors: Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
authors: Ágnes
authors: Á
authors: Kittel
authors: Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, United States of America; Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Bence
authors: B
authors: György
authors: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Éva
authors: É
authors: Pállinger
authors: Department of Immunology, Eötvös Loránd University, Budapest, Hungary.
authors: János
authors: J
authors: Matkó
authors: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
authors: Edit Irén
authors: EI
authors: Buzás
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000032:analysis*; D000067128:Extracellular Vesicles / Q000737:chemistry*; D005677:G(M1) Ganglioside / Q000032:analysis*; D006801:Humans; D019169:Jurkat Cells; D008051:Lipid Bilayers / Q000737:chemistry; D051379:Mice; D011506:Proteins / Q000032:analysis*
chemical_list: D008051:Lipid Bilayers; D011506:Proteins; D005677:G(M1) Ganglioside; D002784:Cholesterol