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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180059	6/6	Gut microbiota	Stool	DG SEC	Tulkens J	2018	87%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 87% (96th 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 Stool Sample origin Control condition Focus vesicles gut bacteria-derived EV 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 SEC Protein markers EV: Proteomics/ Western blot/ LPS/ OmpA/ Limulus Amebocyte Lysate Assay non-EV: Alix/ flotilin-1/ CD9 Proteomics yes EV density (g/ml) 1.141-1.186 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Stool Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10 Highest density fraction 50 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.667 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins OmpA Not detected contaminants Alix/ flotilin-1/ CD9 Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes Particle yield particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV180059	1/6	Gram-negative bacteria	Escherichia coli Nissle 1917	DG SEC	Tulkens J	2018	75%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 75% (96th 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 Escherichia coli 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 SEC Protein markers EV: Limulus Amebocyte Lysate assay/ LPS/ OmpA/ Western blot non-EV: Proteomics no EV density (g/ml) 1.141-1.186 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gram-negative bacteria Sample Type Cell culture supernatant EV-producing cells Escherichia coli Nissle 1917 EV-harvesting Medium Serum free medium Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10 Highest density fraction 50 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.667 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins OmpA/ LPS Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes EM EM-type Immuno-EM EM protein LPS Image type Close-up, Wide-field

	EV180059	2/6	Gut microbiota	Blood plasma	DG SEC	Tulkens J	2018	42%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 42% (74th 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 Blood plasma Sample origin Control condition Focus vesicles LPS-positive bacterial EV 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 SEC Protein markers EV: LPS/ OmpA non-EV: Proteomics no EV density (g/ml) 1.141-1.186 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16,5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No

	EV180059	3/6	Gut microbiota	Blood plasma	SEC	Tulkens J	2018	42%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 42% (74th 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 Blood plasma Sample origin Inflammatory bowel disease Focus vesicles LPS-positive bacterial EV 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 SEC Protein markers EV: LPS/ OmpA non-EV: Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Immuno-EM EM protein LPS Image type Close-up

	EV180059	5/6	Gut microbiota	Blood plasma	SEC	Tulkens J	2018	42%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 42% (74th 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 Blood plasma Sample origin Therapy-induced intestinal mucositis Focus vesicles LPS-positive bacterial EV 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 SEC Protein markers EV: LPS/ OmpA non-EV: Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Immuno-EM EM protein E. coli LPS Image type Close-up

	EV180059	4/6	Gut microbiota	Blood plasma	SEC	Tulkens J	2018	28%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 28% (58th 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 Blood plasma Sample origin HIV Focus vesicles LPS-positive bacterial EV 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 SEC Protein markers EV: LPS/ OmpA non-EV: Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No

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EV-TRACK ID	EV180059
species	Gut microbiota	Gram-negative bacteria	Gut microbiota	Gut microbiota	Gut microbiota	Gut microbiota
sample type	Stool	Cell culture	Blood plasma	Blood plasma	Blood plasma	Blood plasma
cell type	NA	Escherichia coli Nissle 1917	NA	NA	NA	NA
medium	NA	Serum free medium	NA	NA	NA	NA
condition	Control condition	Control condition	Control condition	Inflammatory bowel disease	Therapy-induced intestinal mucositis	HIV
separation protocol	DG SEC	DG SEC	DG SEC	SEC	SEC	SEC
vesicle related term	gut bacteria-derived EV	Escherichia coli	LPS-positive bacterial EV	LPS-positive bacterial EV	LPS-positive bacterial EV	LPS-positive bacterial EV
Exp. nr.	6	1	2	3	5	4
EV-METRIC %	87	75	42	42	42	28