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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140037	1/1	Homo sapiens	NAY	(d)(U)C IAF Total Exosome Isolation	Oksvold MP	2014	38%
Study summary Full title Expression of B-cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells All authors Oksvold MP, Kullmann A, Forfang L, Kierulf B, Li M, Brech A, Vlassov AV, Smeland EB, Neurauter A, Pedersen KW Journal Clin Ther Abstract PURPOSE: Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found (show more...)PURPOSE: Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81(+) and CD63(+) subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation. METHODS: Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81. FINDINGS: Magnetic bead-based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma-derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity. IMPLICATIONS: Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C IAF Total Exosome Isolation Protein markers EV: CD81/ CD63/ MHC2/ CD9 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation Immunoaffinity capture Selected surface protein(s) CD81, CD63 Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ MHC2 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MHC2 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV140037
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C IAF Total Exosome Isolation
Exp. nr.	1
EV-METRIC %	38