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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230298	1/3	Escherichia coli	APEC O1	DG (d)(U)C UF Filtration	Hu R	2020	57%
Study summary Full title Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC). All authors Hu R, Li J, Zhao Y, Lin H, Liang L, Wang M, Liu H, Min Y, Gao Y, Yang M Journal Microb Cell Fact Abstract The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its nu (show more...)The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. (hide) EV-METRIC 57% (91st 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 Other/ Outer membrane vesicles 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 Density gradient (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells APEC O1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Lowest density fraction 10% Highest density fraction 55% Speed (g) 180000 Duration (min) 960 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EM EM-type Transmission-EM/ Scanning-EM Image type Wide-field

	EV230298	2/3	Escherichia coli	APEC O2	DG (d)(U)C UF Filtration	Hu R	2020	57%
Study summary Full title Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC). All authors Hu R, Li J, Zhao Y, Lin H, Liang L, Wang M, Liu H, Min Y, Gao Y, Yang M Journal Microb Cell Fact Abstract The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its nu (show more...)The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. (hide) EV-METRIC 57% (91st 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 Other/ Outer membrane vesicles 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 Density gradient (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells APEC O2 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Lowest density fraction 10% Highest density fraction 55% Speed (g) 180000 Duration (min) 960 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230298	3/3	Escherichia coli	APEC 078	DG (d)(U)C UF Filtration	Hu R	2020	57%
Study summary Full title Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC). All authors Hu R, Li J, Zhao Y, Lin H, Liang L, Wang M, Liu H, Min Y, Gao Y, Yang M Journal Microb Cell Fact Abstract The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its nu (show more...)The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. (hide) EV-METRIC 57% (91st 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 Other/ Outer membrane vesicles 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 Density gradient (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells APEC 078 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Lowest density fraction 10% Highest density fraction 55% Speed (g) 180000 Duration (min) 960 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV230298
species	Escherichia coli
sample type	Cell culture
cell type	APEC O1	APEC O2	APEC 078
condition	Control condition	Control condition	Control condition
separation protocol	Density gradient/ dUC/ Ultrafiltration/ Filtration	Density gradient/ dUC/ Ultrafiltration/ Filtration	Density gradient/ dUC/ Ultrafiltration/ Filtration
Exp. nr.	1	2	3
EV-METRIC %	57	57	57