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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190080	1/3	Homo sapiens	OVCAR5	DG (d)(U)C SEC SEC (non-commercial)	Zaborowski MP	2019	56%
Study summary Full title Effects of high-dose phytoestrogens on circulating cellular microparticles and coagulation function in postmenopausal women. All authors Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, Zappulli V, Maas SLN, Allen RM, Rumde P, György B, Aufiero M, Schweiger MW, Lai CP, Weissleder R, Lee H, Vickers KC, Tannous BA, Breakefield XO. Journal Sci Rep Abstract Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter (show more...)Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models. (hide) EV-METRIC 56% (90th 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 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 DG (d)(U)C SEC SEC (non-commercial) Protein markers EV: CD63 non-EV: Proteomics no EV density (g/ml) 1.11 Show all info Study aim New methodological development/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells OVCAR5 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120101 Wash: volume per pellet (ml) 40 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 120101 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 8% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 200620 Duration (min) 38 Fraction volume (mL) 350 Fraction processing None Size-exclusion chromatography Total column volume (mL) 24 Sample volume/column (mL) 1 Resin type Superdex 200 Other Name other separation method SEC (non-commercial) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Other 1 https://www.ncbi.nlm.nih.gov/pubmed/30943406 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-120 Other particle analysis name(1) Report type EV-concentration

	EV190080	2/3	Homo sapiens	A2780	DG	Zaborowski MP	2019	38%
Study summary Full title Effects of high-dose phytoestrogens on circulating cellular microparticles and coagulation function in postmenopausal women. All authors Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, Zappulli V, Maas SLN, Allen RM, Rumde P, György B, Aufiero M, Schweiger MW, Lai CP, Weissleder R, Lee H, Vickers KC, Tannous BA, Breakefield XO. Journal Sci Rep Abstract Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter (show more...)Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models. (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 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 DG Protein markers EV: CD63 non-EV: Proteomics no EV density (g/ml) Not specified Show all info Study aim New methodological development/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A2780 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 8% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 200620 Duration (min) 38 Fraction volume (mL) 0.35 Fraction processing None 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 CD63 Other 1 https://www.ncbi.nlm.nih.gov/pubmed/30943406 Characterization: Lipid analysis No Other particle analysis name(1) Report type EV-concentration

	EV190080	3/3	Homo sapiens	OVCA433	DG	Zaborowski MP	2019	17%
Study summary Full title Effects of high-dose phytoestrogens on circulating cellular microparticles and coagulation function in postmenopausal women. All authors Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, Zappulli V, Maas SLN, Allen RM, Rumde P, György B, Aufiero M, Schweiger MW, Lai CP, Weissleder R, Lee H, Vickers KC, Tannous BA, Breakefield XO. Journal Sci Rep Abstract Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter (show more...)Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models. (hide) EV-METRIC 17% (54th 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 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 DG Protein markers EV: non-EV: Proteomics no EV density (g/ml) Not specified Show all info Study aim New methodological development/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells OVCA433 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 8% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 200620 Duration (min) 38 Fraction volume (mL) 0.35 Fraction processing None Protein Concentration Method Not determined Characterization: Lipid analysis No Other particle analysis name(1) Report type EV-concentration

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EV-TRACK ID	EV190080
species	Homo sapiens
sample type	Cell culture
cell type	OVCAR5	A2780	OVCA433
condition	Control condition	Control condition	Control condition
separation protocol	DG (d)(U)C SEC SEC (non-commercial)	DG	DG
Exp. nr.	1	2	3
EV-METRIC %	56	38	17