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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210389	1/1	Homo sapiens	Urine	dUC	Liu Z	2018	56%
Study summary Full title Isolation and characterization of human urine extracellular vesicles. All authors Liu Z, Cauvi DM, Bernardino EMA, Lara B, Lizardo RE, Hawisher D, Bickler S, De Maio A Journal Cell Stress Chaperones Abstract Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential (show more...)Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential biomarkers for disease. They can be obtained from a variety of body fluids, particularly urine that is an ideal source because it can be obtained in great quantities, recurrently and with minimal intervention. However, the characterization of urine ECV is challenging because the preparation is usually contaminated with soluble proteins, such as uromodulin (UMOD) or Tamm-Horsfall glycoprotein that forms large extracellular filaments co-sedimenting with ECV. We developed a method to obtain human urine ECV free of UMOD by the addition of ZnSO prior to vesicle isolation by differential centrifugation. Treatment with ZnSO did not affect the size and concentration of the vesicle preparation and preserved the storage of the samples at low temperatures. We did not observe a variation in the number of vesicles isolated during different times of the day or different days between different donors. The glycoprotein pattern of urine ECV was characterized by binding to concanavalin A (Con A) and mass spectroscopy. Several markers were found, including dipeptidyl peptidase IV (CD26), vacuolar protein sorting factor 4A (VPS4A) and dipeptidase 1 (DPEP1), and galectin 3 binding protein (G3-BP). The levels of VPS4A and DPEP1 were similar in ECV preparations obtained from several donors of both sexes. Con A binding pattern and monosaccharide composition were also comparable between subjects. In summary, our method for the isolation of highly pure ECV derived from human urine is likely to help in the use of these vesicles as potential biomarkers. (hide) EV-METRIC 56% (89th 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 Urine Sample origin Control condition Focus vesicles extracellular vesicle 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 dUC Protein markers EV: DPEP1/ CD81/ G3BP/ CD9/ VPS4A non-EV: Tamm-Horsfall protein Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 60 Wash: speed (g) 100000 Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ VPS4A/ DPEP1/ G3BP/ CD81 Not detected contaminants Tamm-Horsfall protein Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 136.9 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.00e+10

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EV-TRACK ID	EV210389
species	Homo sapiens
sample type	Urine
condition	Control condition
separation protocol	dUC
Exp. nr.	1
EV-METRIC %	56