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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230522	3/3	Neisseria gonorrhoeae	DMS 15130	(d)(U)C DG Filtration	Pérez-Cruz C	2015	43%
Study summary Full title Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria. All authors Pérez-Cruz C, Delgado L, López-Iglesias C, Mercade E Journal PLoS One Abstract Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle sec (show more...)Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles. (hide) EV-METRIC 43% (81st 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 Outer-inner membrane vesicles (O-IMVs) 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 Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Neisseria gonorrhoeae Sample Type Cell culture supernatant EV-producing cells DMS 15130 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: speed (g) 40000 Wash: volume per pellet (ml) 50 Wash: time (min) 60 Wash: speed (g) 40000 Density gradient Type Continuous Highest density fraction 45% Sample volume (mL) 4 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction processing None Filtration steps Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Cryo-EM/ Transmission-EM Image type Close-up, Wide-field Report size (nm) 85

	EV230522	1/3	Acinetobacter baumannii	AB41	(d)(U)C Filtration	Pérez-Cruz C	2015	14%
Study summary Full title Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria. All authors Pérez-Cruz C, Delgado L, López-Iglesias C, Mercade E Journal PLoS One Abstract Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle sec (show more...)Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles. (hide) EV-METRIC 14% (44th 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 Outer-inner membrane vesicles (O-IMVs) 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 Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Acinetobacter baumannii Sample Type Cell culture supernatant EV-producing cells AB41 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: speed (g) 40000 Wash: volume per pellet (ml) 50 Wash: time (min) 60 Wash: speed (g) 40000 Filtration steps Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Cryo-EM/ Transmission-EM Image type Close-up, Wide-field Report size (nm) 109

	EV230522	2/3	Pseudomonas aeruginosa	PAO1	(d)(U)C Filtration	Pérez-Cruz C	2015	14%
Study summary Full title Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria. All authors Pérez-Cruz C, Delgado L, López-Iglesias C, Mercade E Journal PLoS One Abstract Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle sec (show more...)Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles. (hide) EV-METRIC 14% (44th 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 Outer-inner membrane vesicles (O-IMVs) 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 Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Pseudomonas aeruginosa Sample Type Cell culture supernatant EV-producing cells PAO1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: speed (g) 40000 Wash: volume per pellet (ml) 50 Wash: time (min) 60 Wash: speed (g) 40000 Filtration steps Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Cryo-EM/ Transmission-EM Image type Close-up, Wide-field Report size (nm) 135

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EV-TRACK ID	EV230522
species	Neisseria gonorrhoeae	Acinetobacter baumannii	Pseudomonas aeruginosa
sample type	Cell culture	Cell culture	Cell culture
cell type	DMS 15130	AB41	PAO1
condition	Control condition	Control condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration	dUC/ Filtration	dUC/ Filtration
Exp. nr.	3	1	2
EV-METRIC %	43	14	14