EV-TRACK

Search > Results

You searched for: 2015 (Year of publication)

Showing 51 - 100 of 784

Results list

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV150011	1/1	Homo sapiens	NAY	(d)(U)C DG	Lazar I	2015	44%
Study summary Full title Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines All authors Lazar I, Clement E, Ducoux-Petit M, Denat L, Soldan V, Dauvillier S, Balor S, Burlet-Schiltz O, Larue L, Muller C, Nieto L Journal Pigm Cell Melanoma R Abstract Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma (show more...)Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma-specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro-migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells' aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment. (hide) EV-METRIC 44% (84th 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 NAY Focus vesicles exosomes 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 (d)(U)C DG Protein markers EV: CD81/ TSG101/ Flotilin1 non-EV: Proteomics yes EV density (g/ml) 1.13-1.19 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Top-down Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Flotilin1/ TSG101 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV150005	2/2	Mus musculus	NAY	(d)(U)C Filtration	Nojima H	2015	44%
Study summary Full title Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate All authors Nojima H, Freeman CM, Schuster RM, Japtok L, Kleuser B, Edwards MJ, Gulbins E, Lentsch AB Journal J Hepatol Abstract BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hep (show more...)BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. METHODS: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. RESULTS: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. CONCLUSIONS: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (hide) EV-METRIC 44% (84th 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 NAY Focus vesicles exosomes 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 (d)(U)C Filtration Protein markers EV: CD81/ TSG101/ CD63 non-EV: Beta-actin/ Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ TSG101 Detected contaminants Cell organelle protein/ Beta-actin Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

	EV150024	1/2	Homo sapiens	NAY	(d)(U)C	Hazan-Halevy I	2015	44%
Study summary Full title Cell-specific uptake of mantle cell lymphoma-derived exosomes by malignant and non-malignant B-lymphocytes All authors Hazan-Halevy I, Rosenblum D, Weinstein S, Bairey O, Raanani P, Peer D Journal Cancer Lett Abstract Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatm (show more...)Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatments are based on chemotherapeutics and new class of drugs (e.g. Ibrutinib(®)), which in most cases ends with tumor resistance and relapse. Therefore, further development of novel therapeutic modalities is needed. Exosomes are natural extracellular vesicles, which play an important role in intercellular communication. The specificity of exosome uptake by different target cells remains unknown. In this study, we observed that MCL exosomes are taken up rapidly and preferentially by MCL cells. Only a minor fraction of exosomes was internalized into T-cell leukemia and bone marrow stroma cell lines, when these cells were co-cultured with MCL cells. Moreover, MCL patients' exosomes were taken up by both healthy and patients' B-lymphocytes with no apparent internalization to T lymphocytes and NK cells. Exosome internalization was not inhibited by specific siRNA against caveolin1 and clathrin but was found to be mediated by a cholesterol-dependent pathway. These findings demonstrate natural specificity of exosomes to B-lymphocytes and ultimately might be used for therapeutic intervention in B cells malignancies. (hide) EV-METRIC 44% (84th 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 NAY Focus vesicles exosomes 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 (d)(U)C Protein markers EV: CD81/ TSG101/ CD63/ CD19 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ TSG101/ CD19 Detected contaminants Calnexin ELISA Detected EV-associated proteins CD19 Flow cytometry specific beads Selected surface protein(s) Yes Characterization: Particle analysis NTA EM EM-type transmission EM/ immune EM EM protein CD81 Image type Close-up

	EV150039	1/1	Homo sapiens	Semen	(d)(U)C DG SEC	Dubois L	2015	44%
Study summary Full title Proteomic profiling of detergent resistant membranes (lipid rafts) of prostasomes All authors Dubois L, Ronquist KK, Ek B, Ronquist G, Larsson A Journal Mol Cell Proteomics Abstract Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular (show more...)Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163. (hide) EV-METRIC 44% (82nd 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 Semen Sample origin NAY Focus vesicles Prostasomes 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 (d)(U)C DG SEC Adj. k-factor 126 (pelleting) Protein markers EV: Clathrin/ Flotilin1/ Flotillin2 non-EV: Proteomics yes EV density (g/ml) 1.13-1.19 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Semen Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 90Ti Pelleting: adjusted k-factor 126.0 Density gradient Lowest density fraction 1 Highest density fraction 2 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ Flotillin2/ Clathrin ELISA Detected EV-associated proteins Flotillin2/ Clathrin Characterization: Particle analysis None

	EV230676	1/1	Escherichia coli	MG1655	(d)(U)C DG Filtration	Kulkarni HM	2015	43%
Study summary Full title Protective role of E. coli outer membrane vesicles against antibiotics. All authors Kulkarni HM, Nagaraj R, Jagannadham MV Journal Microbiol Res Abstract The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective f (show more...)The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics. (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 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 Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells MG1655 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: time(min) 180 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0% Highest density fraction 70% Orientation Top-down Speed (g) 160000 Duration (min) 240 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: speed (g) 160000 Pelleting: adjusted k-factor 7.566 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 80-90 EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-80

	EV230672	2/4	Vibrio cholerae	A1552	(d)(U)C DG Filtration	Sjöström AE	2015	43%
Study summary Full title Membrane vesicle-mediated release of bacterial RNA. All authors Sjöström AE, Sandblad L, Uhlin BE, Wai SN Journal Sci Rep Abstract Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the h (show more...)Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions. (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 AES206 mutant Focus vesicles 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 (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 0 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells A1552 EV-harvesting Medium Serum free medium 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: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 0 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Orientation Top-down Speed (g) 180000 Duration (min) 120 Fraction volume (mL) 0.1-0.3 Fraction processing Centrifugation Pelleting: volume per fraction 0.1-0.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1,70E Pelleting-wash: volume per pellet (mL) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing/ Northern blot Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230672	3/4	Vibrio cholerae	A1552	(d)(U)C DG Filtration	Sjöström AE	2015	43%
Study summary Full title Membrane vesicle-mediated release of bacterial RNA. All authors Sjöström AE, Sandblad L, Uhlin BE, Wai SN Journal Sci Rep Abstract Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the h (show more...)Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions. (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 AES207 mutant Focus vesicles 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 (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 0 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells A1552 EV-harvesting Medium Serum free medium 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: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 0 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Orientation Top-down Speed (g) 180000 Duration (min) 120 Fraction volume (mL) 0.1-0.3 Fraction processing Centrifugation Pelleting: volume per fraction 0.1-0.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1,70E Pelleting-wash: volume per pellet (mL) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing/ Northern blot Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230672	4/4	Vibrio cholerae	A1552	(d)(U)C DG Filtration	Sjöström AE	2015	43%
Study summary Full title Membrane vesicle-mediated release of bacterial RNA. All authors Sjöström AE, Sandblad L, Uhlin BE, Wai SN Journal Sci Rep Abstract Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the h (show more...)Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions. (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 AES208 mutant Focus vesicles 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 (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 0 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells A1552 EV-harvesting Medium Serum free medium 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: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 0 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Orientation Top-down Speed (g) 180000 Duration (min) 120 Fraction volume (mL) 0.1-0.3 Fraction processing Centrifugation Pelleting: volume per fraction 0.1-0.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 1,70E Pelleting-wash: volume per pellet (mL) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type RNA sequencing/ Northern blot Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	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

	EV150001	1/1	Homo sapiens	Urine	(d)(U)C UF	Lozano-Ramos I	2015	43%
Study summary Full title Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples All authors Lozano-Ramos I, Bancu I, Oliveira-Tercero A, Armengol MP, Menezes-Neto A, Del Portillo HA, Lauzurica-Valdemoros R, Borràs FE Journal J Extracell Vesicles Abstract Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this inv (show more...)Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs.When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm-Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species.To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches. (hide) EV-METRIC 43% (77th 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 Urine Sample origin NAY Focus vesicles extracellular 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 (d)(U)C UF Protein markers EV: CD63/ CD9 non-EV: Tamm-Horsfall glycoprotein/ Albumin Proteomics no TEM measurements 80-120 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed No Characterization: Particle analysis NTA EM EM-type cryo EM Image type Close-up, Wide-field Report size (nm) 80-120

	EV150018	2/2	Mus musculus	Brain tissue	(d)(U)C DG IAF	Asai H	2015	43%
Study summary Full title Depletion of microglia and inhibition of exosome synthesis halt tau propagation All authors Asai H, Ikezu S, Tsunoda S, Medalla M, Luebke J, Haydar T, Wolozin B, Butovsky O, Kügler S, Ikezu T Journal Nat Neurosci Abstract Accumulation of pathological tau protein is a major hallmark of Alzheimer's disease. Tau protein spr (show more...)Accumulation of pathological tau protein is a major hallmark of Alzheimer's disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus-based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target. (hide) EV-METRIC 43% (46th 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 Brain tissue Sample origin NAY Focus vesicles exosomes 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 (d)(U)C DG IAF Protein markers EV: AChE non-EV: Proteomics no TEM measurements 106.4+-29.6 Show all info Study aim Function Sample Species Mus musculus Sample Type Brain tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Wash: volume per pellet (ml) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Immunoaffinity capture Selected surface protein(s) Tsg101 Western Blot Detected EV-associated proteins AChE ELISA Detected EV-associated proteins AChE Characterization: Particle analysis EM EM-type immune EM EM protein Tsg101 Image type Close-up Report size (nm) 106.4+-29.6

	EV230674	1/2	Klebsiella pneumoniae	ATCC 43816	(d)(U)C DG Filtration UF	Cahill BK	2015	38%
Study summary Full title Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles. All authors Cahill BK, Seeley KW, Gutel D, Ellis TN Journal Microbiol Res Abstract Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and (show more...)Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane. (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 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 Filtration Ultrafiltration Protein markers EV: GroEL non-EV: RecA Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Klebsiella pneumoniae Sample Type Cell culture supernatant EV-producing cells ATCC 43816 EV-harvesting Medium serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Orientation Bottom-up Speed (g) 100000 Fraction volume (mL) 1 Fraction processing None Filtration steps > 0.45 µm, 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins GroEL Not detected contaminants RecA Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis None

	EV230674	2/2	Klebsiella pneumoniae	ATCC 43816	(d)(U)C DG Filtration UF	Cahill BK	2015	38%
Study summary Full title Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles. All authors Cahill BK, Seeley KW, Gutel D, Ellis TN Journal Microbiol Res Abstract Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and (show more...)Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane. (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 wbbO K.O. 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 Filtration Ultrafiltration Protein markers EV: GroEL non-EV: RecA Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Klebsiella pneumoniae Sample Type Cell culture supernatant EV-producing cells ATCC 43816 EV-harvesting Medium serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Orientation Bottom-up Speed (g) 100000 Fraction volume (mL) 1 Fraction processing None Filtration steps > 0.45 µm, 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins GroEL Not detected contaminants RecA Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis None

	EV230656	3/21	Vibrio cholerae	C6706	NA	Rompikuntal PK	2015	38%
Study summary Full title Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae. All authors Rompikuntal PK, Vdovikova S, Duperthuy M, Johnson TL, Åhlund M, Lundmark R, Oscarsson J, Sandkvist M, Uhlin BE, Wai SN Journal PLoS One Abstract Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during no (show more...)Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. (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 Outer membrane vesicle (OMV) 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 NA Protein markers EV: PrtV/ OmpU/ none non-EV: Crp/ none/ nonen Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells C6706 EV-harvesting Medium Serum free medium Separation Method Other Name other separation method NA Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins PrtV/ OmpU Not detected EV-associated proteins none Detected contaminants none Not detected contaminants Crp Other 2 SDS-PAGE Detected EV-associated proteins none Not detected EV-associated proteins none Detected contaminants none Not detected contaminants nonen Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 105 EV concentration Yes EM EM-type Immuno-EM Image type Wide-field

	EV230656	5/21	Vibrio cholerae	C6706	NA	Rompikuntal PK	2015	38%
Study summary Full title Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae. All authors Rompikuntal PK, Vdovikova S, Duperthuy M, Johnson TL, Åhlund M, Lundmark R, Oscarsson J, Sandkvist M, Uhlin BE, Wai SN Journal PLoS One Abstract Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during no (show more...)Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. (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 delprtV Focus vesicles Outer membrane vesicle (OMV) 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 NA Protein markers EV: OmpU/ PrtV/ none non-EV: Crp/ none Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells C6706 EV-harvesting Medium Serum free medium Separation Method Other Name other separation method NA Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins OmpU Not detected EV-associated proteins PrtV Detected contaminants none Not detected contaminants Crp Other 1 SDS-PAGE Detected EV-associated proteins none Not detected EV-associated proteins none Detected contaminants none Not detected contaminants none Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 105 EV concentration Yes EM EM-type Immuno-EM Image type Wide-field

	EV150007	2/10	Homo sapiens	Blood plasma	(d)(U)C UF qEV	Lobb RJ	2015	38%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 38% (70th 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 NAY Focus vesicles exosomes 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 (d)(U)C UF qEV Protein markers EV: Flotilin1 non-EV: Albumin/ Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1 Detected contaminants Cell organelle protein/ Albumin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV150007	9/10	Homo sapiens	Blood plasma	(d)(U)C ExoQuick Filtration	Lobb RJ	2015	38%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 38% (70th 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 NAY Focus vesicles exosomes 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 (d)(U)C ExoQuick Filtration Protein markers EV: Flotilin1 non-EV: Albumin/ Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1 Detected contaminants Cell organelle protein/ Albumin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Close-up

	EV150007	10/10	Homo sapiens	Blood plasma	(d)(U)C Exo-spin Filtration	Lobb RJ	2015	38%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 38% (70th 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 NAY Focus vesicles exosomes 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 (d)(U)C Exo-spin Filtration Protein markers EV: Flotilin1 non-EV: Albumin/ Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit Exo-spin Other Name other separation method Exo-spin Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1 Detected contaminants Cell organelle protein/ Albumin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV150005	1/2	Mus musculus	Serum	ExoQuick	Nojima H	2015	38%
Study summary Full title Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate All authors Nojima H, Freeman CM, Schuster RM, Japtok L, Kleuser B, Edwards MJ, Gulbins E, Lentsch AB Journal J Hepatol Abstract BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hep (show more...)BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. METHODS: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. RESULTS: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. CONCLUSIONS: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (hide) EV-METRIC 38% (82nd 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 Serum Sample origin NAY Focus vesicles exosomes 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 ExoQuick Protein markers EV: CD81/ TSG101/ CD63 non-EV: Beta-actin/ Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ TSG101 Detected contaminants Cell organelle protein/ Beta-actin Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV150004	2/2	Homo sapiens	NAY	Filtration UF qEV	Clark DJ	2015	38%
Study summary Full title Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis All authors Clark DJ, Fondrie WE, Liao Z, Hanson PI, Fulton A, Mao L, Yang AJ Journal Anal Chem Abstract Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into t (show more...)Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as true exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression. (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 NAY Focus vesicles exosomes 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 Filtration UF qEV Protein markers EV: Alix/ CD63 non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63 Characterization: Particle analysis None

	EV230644	1/1	Tannerella forsythia	ATCC 43037	(d)(U)C Filtration	Friedrich V	2015	34%
Study summary Full title Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence. All authors Friedrich V, Gruber C, Nimeth I, Pabinger S, Sekot G, Posch G, Altmann F, Messner P, Andrukhov O, Schäffer C Journal Mol Oral Microbiol Abstract Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown (show more...)Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential. (hide) EV-METRIC 34% (78th 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 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 Filtration Protein markers EV: TfsA-GP/ TfsB-GP non-EV: None Proteomics yes Show all info Study aim Function/Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Tannerella forsythia Sample Type Cell culture supernatant EV-producing cells ATCC 43037 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 25 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method 2-D Quant kit Western Blot Detected EV-associated proteins TfsA-GP/ TfsB-GP Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV230666	1/5	Aggregatibacter actinomycetemcomitans	Strain 173	(d)(U)C DG Filtration	Kieselbach T	2015	33%
Study summary Full title Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles. All authors Kieselbach T, Zijnge V, Granström E, Oscarsson J Journal PLoS One Abstract Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive fo (show more...)Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. (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 Control condition 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 Filtration Adj. k-factor 1 (pelleting) / 1 (washing) Protein markers EV: GroEL non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Aggregatibacter actinomycetemcomitans Sample Type Cell culture supernatant EV-producing cells Strain 173 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 85000 Pelleting: adjusted k-factor 1,52E Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 85000 Wash: adjusted k-factor 1,52E Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 10% Highest density fraction 45% Total gradient volume, incl. sample (mL) 4.170 Sample volume (mL) 0.150 Orientation Bottom-up Speed (g) 180000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Centrifugation Pelleting: speed (g) 16000 Pelleting: adjusted k-factor 9,55E Pelleting-wash: speed (g) JA-18.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Spectrophotometry Western Blot Detected EV-associated proteins GroEL Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Atomic force microscopy Image type Wide-field

	EV230662	2/6	Escherichia coli	BL21(DE3)	(d)(U)C Filtration	Kumar S	2015	33%
Study summary Full title Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence. All authors Kumar S, Mittal E, Deore S, Kumar A, Rahman A, Krishnasastry MV Journal Front Cell Infect Microbiol Abstract The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was r (show more...)The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as "hemolysin" which was re-annotated as 2'-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms. (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 pTlyA transformed 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 Filtration Protein markers EV: TlyA/ β-lactamase non-EV: None Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells BL21(DE3) Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-100 Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins TlyA/ β-lactamase Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ immuno-EM EM protein TlyA Image type Wide-field

	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

	EV230656	2/21	Vibrio cholerae	C6706	(d)(U)C Filtration	Rompikuntal PK	2015	33%
Study summary Full title Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae. All authors Rompikuntal PK, Vdovikova S, Duperthuy M, Johnson TL, Åhlund M, Lundmark R, Oscarsson J, Sandkvist M, Uhlin BE, Wai SN Journal PLoS One Abstract Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during no (show more...)Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. (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 Control condition Focus vesicles Outer membrane vesicle (OMV) 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: OmpU/ PrtV/ none non-EV: Crp/ none Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells C6706 EV-harvesting Medium Serum free medium 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: rotor type Type 45 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins OmpU/ PrtV Not detected contaminants Crp Other 2 SDS-PAGE Detected EV-associated proteins none Not detected EV-associated proteins none Detected contaminants none Not detected contaminants none Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230656	4/21	Vibrio cholerae	C6706	(d)(U)C Filtration	Rompikuntal PK	2015	33%
Study summary Full title Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae. All authors Rompikuntal PK, Vdovikova S, Duperthuy M, Johnson TL, Åhlund M, Lundmark R, Oscarsson J, Sandkvist M, Uhlin BE, Wai SN Journal PLoS One Abstract Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during no (show more...)Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. (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 delprtV Focus vesicles Outer membrane vesicle (OMV) 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: OmpU/ PrtV/ none non-EV: Crp/ none Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells C6706 EV-harvesting Medium Serum free medium 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: rotor type Type 45 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins OmpU Not detected EV-associated proteins PrtV Detected contaminants none Not detected contaminants Crp Other 1 SDS-PAGE Detected EV-associated proteins none Not detected EV-associated proteins none Detected contaminants none Not detected contaminants none Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230656	8/21	Vibrio cholerae	C6706	(d)(U)C Filtration	Rompikuntal PK	2015	33%
Study summary Full title Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae. All authors Rompikuntal PK, Vdovikova S, Duperthuy M, Johnson TL, Åhlund M, Lundmark R, Oscarsson J, Sandkvist M, Uhlin BE, Wai SN Journal PLoS One Abstract Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during no (show more...)Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. (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 delpkd Focus vesicles Outer membrane vesicle (OMV) 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: OmpU/ PrtV/ none non-EV: none Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Vibrio cholerae Sample Type Cell culture supernatant EV-producing cells C6706 EV-harvesting Medium Serum free medium 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: rotor type Type 45 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins OmpU Not detected EV-associated proteins PrtV Detected contaminants none Not detected contaminants none Other 1 SDS-PAGE Detected EV-associated proteins none Not detected EV-associated proteins none Detected contaminants none Not detected contaminants none Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230635	1/2	Porphyromonas gingivalis	ATCC 33277	(d)(U)C Filtration	Bai D	2015	33%
Study summary Full title Immunoreactive antigens recognized in serum samples from mice intranasally immunized with Porphyromonas gingivalis outer membrane vesicles. All authors Bai D, Nakao R, Ito A, Uematsu H, Senpuku H Journal Pathog Dis Abstract Outer membrane vesicles (OMVs) of the periodontopathic bacterium Porphyromonas gingivalis contain a (show more...)Outer membrane vesicles (OMVs) of the periodontopathic bacterium Porphyromonas gingivalis contain a wide range of virulence factors including lipopolysaccharide (LPS), fimbriae and gingipains. We have recently reported strong immunogenicity of OMVs using an intranasal vaccine mouse model. In the present study, we performed sub-immunoproteome analysis of OMV-immunized mouse serum samples from six different mice in order to identify immunodominant antigens. The combination of two-dimensional (2D) gel electrophoresis and mass spectrometry analysis identified OMV proteins of 53 spots on a 2D map, and it was notable that OMV proteins were largely distributed within a low pH range, in marked contrast to the ubiquitous distribution of outer membrane proteins. Western blot using the six serum samples after 2D electrophoresis revealed that all showed immunoreactivity to some diffuse signals at extremely low pH, which was similar to the distribution of immunoreactive signals when the A-LPS antibody was used. Mass spectrometry analysis also demonstrated that the signals corresponded to a wide range of virulence factors including A-LPS-modified proteins such as gingipains. Absorption of serum with LPS resulted in a dramatic reduction of immmunoreactivity. We conclude that LPS and A-LPS-modified proteins in OMVs carry immunodominant determinants and eventually elicit P. gingivalis-specific antibodies in mice. (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 Control condition 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 Filtration Protein markers EV: RgpA/ FimA/ Mfa1 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches)/vaccine development Sample Species Porphyromonas gingivalis Sample Type Cell culture supernatant EV-producing cells ATCC 33277 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) 180 Pelleting: rotor type Type 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins RgpA/ FimA/ Mfa1 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 50-80

	EV230629	1/9	Haemophilus influenzae	Rd KW20	(d)(U)C Filtration	Roier S	2015	33%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (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 Control condition 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 Filtration Protein markers EV: None non-EV: RpoA Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells Rd KW20 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Density gradient Type Discontinuous Number of initial discontinuous layers 8 Lowest density fraction 20% Highest density fraction 50% Total gradient volume, incl. sample (mL) 9.8 + sample Sample volume (mL) not spec Orientation top-down Speed (g) 150000 Duration (min) 1020 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 89

	EV230629	3/9	Haemophilus influenzae	Hib strain Eagan	(d)(U)C Filtration	Roier S	2015	33%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (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 Control condition 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 Filtration Protein markers EV: None non-EV: RpoA Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells Hib strain Eagan EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Density gradient Type Discontinuous Number of initial discontinuous layers 8 Lowest density fraction 20% Highest density fraction 50% Total gradient volume, incl. sample (mL) 9.8 + sample Sample volume (mL) not spec Orientation top-down Speed (g) 150000 Duration (min) 1020 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 152

	EV230629	5/9	Haemophilus influenzae	NTHi 2019-R	(d)(U)C Filtration	Roier S	2015	33%
Study summary Full title A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles. All authors Roier S, Blume T, Klug L, Wagner GE, Elhenawy W, Zangger K, Prassl R, Reidl J, Daum G, Feldman MF, Schild S Journal Int J Med Microbiol Abstract Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released fro (show more...)Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections. (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 Control condition 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 Filtration Protein markers EV: None non-EV: RpoA Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches)/ vaccine development Sample Species Haemophilus influenzae Sample Type Cell culture supernatant EV-producing cells NTHi 2019-R EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Density gradient Type Discontinuous Number of initial discontinuous layers 8 Lowest density fraction 20% Highest density fraction 50% Total gradient volume, incl. sample (mL) 9.8 + sample Sample volume (mL) not spec Orientation top-down Speed (g) 150000 Duration (min) 1020 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 240 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 144000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per mL/OD490 Western Blot Not detected contaminants RpoA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 82

	EV230618	2/3	Vibrio tasmaniensis	LGP32	(d)(U)C DG Filtration	Vanhove AS	2015	33%
Study summary Full title Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells. All authors Vanhove AS, Duperthuy M, Charrière GM, Le Roux F, Goudenège D, Gourbal B, Kieffer-Jaquinod S, Couté Y, Wai SN, Destoumieux-Garzón D Journal Environ Microbiol Abstract Vibrio tasmaniensis LGP32, a facultative intracellular pathogen of oyster haemocytes, was shown (show more...)Vibrio tasmaniensis LGP32, a facultative intracellular pathogen of oyster haemocytes, was shown here to release outer membrane vesicles (OMVs) both in the extracellular milieu and inside haemocytes. Intracellular release of OMVs occurred inside phagosomes of intact haemocytes having phagocytosed few vibrios as well as in damaged haemocytes containing large vacuoles heavily loaded with LGP32. The OMV proteome of LGP32 was shown to be rich in hydrolases (25%) including potential virulence factors such as proteases, lipases, phospholipases, haemolysins and nucleases. One major caseinase/gelatinase named Vsp for vesicular serine protease was found to be specifically secreted through OMVs in which it is enclosed. Vsp was shown to participate in the virulence phenotype of LGP32 in oyster experimental infections. Finally, OMVs were highly protective against antimicrobial peptides, increasing the minimal inhibitory concentration of polymyxin B by 16-fold. Protection was conferred by OMV titration of polymyxin B but did not depend on the activity of Vsp or another OMV-associated protease. Altogether, our results show that OMVs contribute to the pathogenesis of LGP32, being able to deliver virulence factors to host immune cells and conferring protection against antimicrobial peptides. (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 Control condition 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 Filtration Protein markers EV: OmpU non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Vibrio tasmaniensis Sample Type Cell culture supernatant EV-producing cells LGP32 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Density gradient Orientation top-down Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins OmpU Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220212	1/2	Homo sapiens	Fibrocytes	(d)(U)C	Geiger A	2015	33%
Study summary Full title Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice. All authors Geiger A, Walker A, Nissen E Journal Biochem Biophys Res Commun Abstract Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respon (show more...)Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respond to current treatment strategies. This study was addressed to evaluate the therapeutic potential of exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling. The exosomes released from cells sequentially stimulated with platelet-derived growth factor-BB and transforming growth factor-β1, in the presence of fibroblast growth factor 2, did not show potential immunogenicity. These exosomes exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo. Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-126, miR-130a, miR-132) and anti-inflammatory (miR124a, miR-125b) microRNAs, and a microRNA regulating collagen deposition (miR-21). This proof-of-concept study demonstrates the feasibility of the use of fibrocytes-derived exosomes for the treatment of diabetic ulcers. (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 PDGF-BB and TGF-stimulated Focus vesicles exosome 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 Protein markers EV: Flotillin1/ TSG101/ actin/ CD9/ CD63/ CD81/ MHC1/ MHC2/ CD80/ CD86 non-EV: GM130/ calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Fibrocytes EV-harvesting Medium EV-depleted medium Cell count 0 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) number of particles per million cells Western Blot Detected EV-associated proteins Flotillin-1/ TSG101/ actin Not detected contaminants GM130/ calnexin Flow cytometry aspecific beads Detected EV-associated proteins CD9/ CD63/ CD81/ MHC1/ MHC2/ CD80/ CD86 Flow cytometry specific beads Selected surface protein(s) CD9/ CD63/ CD81/ MHC1/ MHC2/ CD80/ CD86 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 89 EV concentration Yes Particle yield number of particles per million cells: 5.90e+8 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 50-100

	EV220212	2/2	Homo sapiens	Fibrocytes	(d)(U)C	Geiger A	2015	33%
Study summary Full title Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice. All authors Geiger A, Walker A, Nissen E Journal Biochem Biophys Res Commun Abstract Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respon (show more...)Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respond to current treatment strategies. This study was addressed to evaluate the therapeutic potential of exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling. The exosomes released from cells sequentially stimulated with platelet-derived growth factor-BB and transforming growth factor-β1, in the presence of fibroblast growth factor 2, did not show potential immunogenicity. These exosomes exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo. Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-126, miR-130a, miR-132) and anti-inflammatory (miR124a, miR-125b) microRNAs, and a microRNA regulating collagen deposition (miR-21). This proof-of-concept study demonstrates the feasibility of the use of fibrocytes-derived exosomes for the treatment of diabetic ulcers. (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 Control condition Focus vesicles exosome 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 Protein markers EV: Flotillin1/ TSG101/ actin/ CD9/ CD63/ CD81 non-EV: GM130/ calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Fibrocytes EV-harvesting Medium EV-depleted medium Cell count 0 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) number of particles per million cells Western Blot Detected EV-associated proteins Flotillin-1/ TSG101/ actin Not detected contaminants GM130/ calnexin Flow cytometry aspecific beads Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Selected surface protein(s) CD9/ CD63/ CD81/ MHC1/ MHC2/ CD80/ CD86 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 81 EV concentration Yes Particle yield number of particles per million cells: 3.40e+8 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 50-100

	EV220075	1/2	Homo sapiens	primary peripheral blood mononuclear cells	DG	Roth WW	2015	33%
Study summary Full title Micro RNA in Exosomes from HIV-Infected Macrophages. All authors Roth WW, Huang MB, Addae Konadu K, Powell MD, Bond VC Journal Int J Environ Res Public Health Abstract Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and protei (show more...)Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients. (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 Control condition Focus vesicles exosome 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 Protein markers EV: CD45/ CD63/ CD9 non-EV: HIV particle Nef/ HIV particle p24 Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary peripheral blood mononuclear cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 mL Sample volume (mL) 1mL Orientation Top-down Rotor type SW 40 Ti Speed (g) 250000 Duration (min) 120 Fraction volume (mL) 1 Fraction processing Centrifugation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD45 Detected contaminants HIV particle Nef/ HIV particle p24 ELISA Detected EV-associated proteins CD63/ CD45/ CD9 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220075	2/2	Homo sapiens	primary peripheral blood mononuclear cells	DG	Roth WW	2015	33%
Study summary Full title Micro RNA in Exosomes from HIV-Infected Macrophages. All authors Roth WW, Huang MB, Addae Konadu K, Powell MD, Bond VC Journal Int J Environ Res Public Health Abstract Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and protei (show more...)Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients. (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 HIV-1 BAL infected Focus vesicles exosome 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 Protein markers EV: CD45/ CD63/ CD9 non-EV: HIV particle Nef/ HIV particle p24 Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary peripheral blood mononuclear cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 mL Sample volume (mL) 1mL Orientation Top-down Rotor type SW 40 Ti Speed (g) 250000 Duration (min) 120 Fraction volume (mL) 1 Fraction processing Centrifugation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD45 Detected contaminants HIV particle Nef/ HIV particle p24 ELISA Detected EV-associated proteins CD63/ CD9/ CD45 Detected contaminants HIV particle Nef/ HIV particle p24 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220053	1/7	Homo sapiens	HUVEC	DG Filtration dUC	Zhu X	2015	33%
Study summary Full title IFITM3-containing exosome as a novel mediator for anti-viral response in dengue virus infection. All authors Zhu X, He Z, Yuan J, Wen W, Huang X, Hu Y, Lin C, Pan J, Li R, Deng H, Liao S, Zhou R, Wu J, Li J, Li M Journal Cell Microbiol Abstract Interferon-inducible transmembrane proteins 1, 2 and 3 (IFITM1, IFITM2 and IFITM3) have recently bee (show more...)Interferon-inducible transmembrane proteins 1, 2 and 3 (IFITM1, IFITM2 and IFITM3) have recently been identified as potent antiviral effectors that function to suppress the entry of a broad range of enveloped viruses and modulate cellular tropism independent of viral receptor expression. However, the antiviral effect and mechanisms of IFITMs in response to viral infections remain incompletely understood and characterized. In this work, we focused our investigation on the function of the extracellular IFITM3 protein. In cell models of DENV-2 infection, we found that IFITM3 contributed to both the baseline and interferon-induced inhibition of DENV entry. Most importantly, our study for the first time demonstrated the presence of IFITM-containing exosome in the extracellular environment, and identified an ability of cellular exosome to intercellularly deliver IFITM3 and thus transmit its antiviral effect from infected to non-infected cells. Thus, our findings provide new insights in the basic mechanisms underlying the actions of IFITM3, which might lead to future development of exosome-mediated anti-viral strategies using IFITM3 as a therapeutic agent. Conceivably, variations in the basal and inducible levels of IFITMs, as well as in intracellular and extracellular levels of IFITMs, might predict the severity of dengue virus infections among individuals or across species. (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 Control condition Focus vesicles exosome 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 Filtration dUC Protein markers EV: IFITM3/ CD63/ Flotillin2 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0.25M Highest density fraction 2.5M Sample volume (mL) 11 Orientation Bottom-up Speed (g) 150000 Duration (min) 960 Fraction processing Centrifugation Pelleting: duration (min) 90 Pelleting: speed (g) 150000 Pelleting-wash: duration (min) 90 Filtration steps 0.22µm or 0.2µm Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ IFITM3/ Flotillin2 Not detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

	EV220053	2/7	Homo sapiens	HUVEC	DG Filtration dUC	Zhu X	2015	33%
Study summary Full title IFITM3-containing exosome as a novel mediator for anti-viral response in dengue virus infection. All authors Zhu X, He Z, Yuan J, Wen W, Huang X, Hu Y, Lin C, Pan J, Li R, Deng H, Liao S, Zhou R, Wu J, Li J, Li M Journal Cell Microbiol Abstract Interferon-inducible transmembrane proteins 1, 2 and 3 (IFITM1, IFITM2 and IFITM3) have recently bee (show more...)Interferon-inducible transmembrane proteins 1, 2 and 3 (IFITM1, IFITM2 and IFITM3) have recently been identified as potent antiviral effectors that function to suppress the entry of a broad range of enveloped viruses and modulate cellular tropism independent of viral receptor expression. However, the antiviral effect and mechanisms of IFITMs in response to viral infections remain incompletely understood and characterized. In this work, we focused our investigation on the function of the extracellular IFITM3 protein. In cell models of DENV-2 infection, we found that IFITM3 contributed to both the baseline and interferon-induced inhibition of DENV entry. Most importantly, our study for the first time demonstrated the presence of IFITM-containing exosome in the extracellular environment, and identified an ability of cellular exosome to intercellularly deliver IFITM3 and thus transmit its antiviral effect from infected to non-infected cells. Thus, our findings provide new insights in the basic mechanisms underlying the actions of IFITM3, which might lead to future development of exosome-mediated anti-viral strategies using IFITM3 as a therapeutic agent. Conceivably, variations in the basal and inducible levels of IFITMs, as well as in intracellular and extracellular levels of IFITMs, might predict the severity of dengue virus infections among individuals or across species. (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 non-targeting control siRNA-transfected Focus vesicles exosome

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data

Study summary

Study data