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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230658	1/4	Lysobacter	VKM B-1576	(d)(U)C DG	Kudryakova IV	2015	33%
Study summary Full title Biogenesis of Lysobacter sp. XL1 vesicles. All authors Kudryakova IV, Suzina NE, Vasilyeva NV Journal FEMS Microbiol Lett Abstract The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracell (show more...)The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained. (hide) EV-METRIC 33% (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 Cell culture supernatant Sample origin 30% sucrose fraction Focus vesicles Outer membrane 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 (Differential) (ultra)centrifugation Density gradient Adj. k-factor 0 (pelleting) / 0 (washing) Protein markers EV: L5 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Lysobacter Sample Type Cell culture supernatant EV-producing cells VKM B-1576 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 115000 Pelleting: adjusted k-factor TDB Wash: time (min) 120 Wash: speed (g) 115000 Wash: adjusted k-factor TDB Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 25% Highest density fraction 55% Sample volume (mL) 2 Orientation Top-down Speed (g) 106500 Duration (min) 720 Fraction processing Centrifugation Pelleting: volume per fraction 30 Pelleting: speed (g) 115000 Pelleting: adjusted k-factor TDB Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins L5 Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Immuno-EM Image type Wide-field Report size (nm) 30-80

	EV230658	2/4	Lysobacter	VKM B-1576	(d)(U)C DG	Kudryakova IV	2015	33%
Study summary Full title Biogenesis of Lysobacter sp. XL1 vesicles. All authors Kudryakova IV, Suzina NE, Vasilyeva NV Journal FEMS Microbiol Lett Abstract The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracell (show more...)The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained. (hide) EV-METRIC 33% (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 Cell culture supernatant Sample origin 35% sucrose fraction Focus vesicles Outer membrane 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 (Differential) (ultra)centrifugation Density gradient Adj. k-factor 0 (pelleting) / 0 (washing) Protein markers EV: L5 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Lysobacter Sample Type Cell culture supernatant EV-producing cells VKM B-1576 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 115000 Pelleting: adjusted k-factor TDB Wash: time (min) 120 Wash: speed (g) 115000 Wash: adjusted k-factor TDB Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 25% Highest density fraction 55% Sample volume (mL) 2 Orientation Top-down Speed (g) 106500 Duration (min) 720 Fraction processing Centrifugation Pelleting: volume per fraction 30 Pelleting: speed (g) 115000 Pelleting: adjusted k-factor TDB Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Not detected EV-associated proteins L5 Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Immuno-EM Image type Wide-field Report size (nm) 65-100

	EV230658	3/4	Lysobacter	VKM B-1576	(d)(U)C DG	Kudryakova IV	2015	33%
Study summary Full title Biogenesis of Lysobacter sp. XL1 vesicles. All authors Kudryakova IV, Suzina NE, Vasilyeva NV Journal FEMS Microbiol Lett Abstract The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracell (show more...)The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained. (hide) EV-METRIC 33% (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 Cell culture supernatant Sample origin 40% sucrose fraction Focus vesicles Outer membrane 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 (Differential) (ultra)centrifugation Density gradient Adj. k-factor 0 (pelleting) / 0 (washing) Protein markers EV: L5 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Lysobacter Sample Type Cell culture supernatant EV-producing cells VKM B-1576 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 115000 Pelleting: adjusted k-factor TDB Wash: time (min) 120 Wash: speed (g) 115000 Wash: adjusted k-factor TDB Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 25% Highest density fraction 55% Sample volume (mL) 2 Orientation Top-down Speed (g) 106500 Duration (min) 720 Fraction processing Centrifugation Pelleting: volume per fraction 30 Pelleting: speed (g) 115000 Pelleting: adjusted k-factor TDB Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) per milliliter of starting sample Western Blot Not detected EV-associated proteins L5 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Immuno-EM Image type Wide-field Report size (nm) 65-100

	EV230658	4/4	Lysobacter	VKM B-1576	(d)(U)C DG	Kudryakova IV	2015	33%
Study summary Full title Biogenesis of Lysobacter sp. XL1 vesicles. All authors Kudryakova IV, Suzina NE, Vasilyeva NV Journal FEMS Microbiol Lett Abstract The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracell (show more...)The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained. (hide) EV-METRIC 33% (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 Cell culture supernatant Sample origin 45% sucrose fraction Focus vesicles Outer membrane 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 (Differential) (ultra)centrifugation Density gradient Adj. k-factor 0 (pelleting) / 0 (washing) Protein markers EV: L5 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Lysobacter Sample Type Cell culture supernatant EV-producing cells VKM B-1576 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 115000 Pelleting: adjusted k-factor TDB Wash: time (min) 120 Wash: speed (g) 115000 Wash: adjusted k-factor TDB Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 25% Highest density fraction 55% Sample volume (mL) 2 Orientation Top-down Speed (g) 106500 Duration (min) 720 Fraction processing Centrifugation Pelleting: volume per fraction 30 Pelleting: speed (g) 115000 Pelleting: adjusted k-factor TDB Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Not detected EV-associated proteins L5 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Immuno-EM Image type Wide-field Report size (nm) 65-100

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EV-TRACK ID	EV230658
species	Lysobacter
sample type	Cell culture
cell type	VKM B-1576
condition	30% sucrose fraction	35% sucrose fraction	40% sucrose fraction	45% sucrose fraction
separation protocol	dUC/ Density gradient	dUC/ Density gradient	dUC/ Density gradient	dUC/ Density gradient
Exp. nr.	1	2	3	4
EV-METRIC %	33	33	33	33