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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200005	2/6	Homo sapiens	Serum	(d)(U)C Filtration qEV	Tzaridis, Theophilos	2021	75%
Study summary Full title Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications All authors Theophilos Tzaridis, Daniel Bachurski, Shu Liu, Kristin Surmann, Felix Babatz, Manuela Gesell Salazar, Uwe Völker, Michael Hallek, Ulrich Herrlinger, Ina Vorberg, Christoph Coch, Katrin S Reiners, Gunther Hartmann Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable s (show more...)Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma. (hide) EV-METRIC 75% (98th 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 Control condition Focus vesicles extracellular 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 (d)(U)C Filtration qEV Protein markers EV: TSG101/ Flotillin1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ TSG101 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 109 EV concentration Yes Particle yield particles/ml;Yes, other: 5.50E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200005	3/6	Homo sapiens	Serum	(d)(U)C Filtration	Tzaridis, Theophilos	2021	75%
Study summary Full title Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications All authors Theophilos Tzaridis, Daniel Bachurski, Shu Liu, Kristin Surmann, Felix Babatz, Manuela Gesell Salazar, Uwe Völker, Michael Hallek, Ulrich Herrlinger, Ina Vorberg, Christoph Coch, Katrin S Reiners, Gunther Hartmann Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable s (show more...)Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma. (hide) EV-METRIC 75% (98th 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 Control condition Focus vesicles extracellular 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 (d)(U)C Filtration Protein markers EV: TSG101/ Flotillin1/ not done non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ TSG101 Detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins not done Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 107 Particle yield NA NA EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200005	4/6	Homo sapiens	Serum	(d)(U)C Filtration qEV UF	Tzaridis, Theophilos	2021	75%
Study summary Full title Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications All authors Theophilos Tzaridis, Daniel Bachurski, Shu Liu, Kristin Surmann, Felix Babatz, Manuela Gesell Salazar, Uwe Völker, Michael Hallek, Ulrich Herrlinger, Ina Vorberg, Christoph Coch, Katrin S Reiners, Gunther Hartmann Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable s (show more...)Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma. (hide) EV-METRIC 75% (98th 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 Control condition Focus vesicles extracellular 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 (d)(U)C Filtration qEV UF Protein markers EV: TSG101/ Flotillin1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ TSG101 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD63/ CD9 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 104 EV concentration Yes Particle yield particles/ml;Yes, other: 3.50E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200005	5/6	Homo sapiens	Serum	(d)(U)C Filtration qEV	Tzaridis, Theophilos	2021	75%
Study summary Full title Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications All authors Theophilos Tzaridis, Daniel Bachurski, Shu Liu, Kristin Surmann, Felix Babatz, Manuela Gesell Salazar, Uwe Völker, Michael Hallek, Ulrich Herrlinger, Ina Vorberg, Christoph Coch, Katrin S Reiners, Gunther Hartmann Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable s (show more...)Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma. (hide) EV-METRIC 75% (98th 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 Control condition Focus vesicles extracellular 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 (d)(U)C Filtration qEV Protein markers EV: TSG101/ Flotillin1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum 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) 105 Pelleting: rotor type TLA-55 Pelleting: speed (g) 110000 Filtration steps 0.45µm > x > 0.22µm, Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ TSG101 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 119 EV concentration Yes Particle yield particles/ml;Yes, other: 4.20E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200005	6/6	Homo sapiens	Serum	(d)(U)C Filtration qEV	Tzaridis, Theophilos	2021	75%
Study summary Full title Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications All authors Theophilos Tzaridis, Daniel Bachurski, Shu Liu, Kristin Surmann, Felix Babatz, Manuela Gesell Salazar, Uwe Völker, Michael Hallek, Ulrich Herrlinger, Ina Vorberg, Christoph Coch, Katrin S Reiners, Gunther Hartmann Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable s (show more...)Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma. (hide) EV-METRIC 75% (98th 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 Control condition Focus vesicles extracellular 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 (d)(U)C Filtration qEV Protein markers EV: TSG101/ Flotillin1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101 Not detected EV-associated proteins Flotillin1 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 155 EV concentration Yes Particle yield particles/ml;Yes, other: 3.20E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210044	1/1	Homo sapiens	Serum	qEV	Newman, Lauren	2021	71%
Study summary Full title Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses All authors Lauren A. Newman, Alia Fahmy, Michael J. Sorich, Oliver G. Best, Andrew Rowland, Zivile Useckaite Journal Cells Abstract Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human bl (show more...)Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a ‘liquid biopsy’ to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease. (hide) EV-METRIC 71% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular 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 qEV Protein markers EV: CD81/ ASGR1/ CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Flow cytometry Type of Flow cytometry CytoFlex S Hardware adaptation to ~100nm EV's 1. Filter configuration as per Beckman Coulter website 2. Violet filter used for small particle detection 3. Calibration beads were used to determine particle size gating strategy 4. Callibration beads used Megamix Plus SSC and Megaamix Plus FSC Calibration bead size 0.1-0.9 Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ASGR1 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 85.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.25E+11 Particle analysis: flow cytometry Flow cytometer type CytoFlex S Hardware adjustment 1. Laser configuration to detect small particles, violet laser use, hardware set up according to Beckman Coulter website 2. Use of MegaMix beads to determine particle size for gating purposes 3. MegaMix plus beads were used: MegaMix Plus SSC and MegaMix Plus Forward FSC Calibration bead size 0.1-0.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210020	1/9	Homo sapiens	MM6	(d)(U)C Filtration DG	Veerman, Rosanne	2021	71%
Study summary Full title Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin All authors Rosanne E. Veerman, Loes Teeuwen, Paulo Czarnewski, Gözde Güclüler Akpinar, AnnSofi Sandberg, Xiaofang Cao, Maria Pernemalm, Lukas M. Orre, Susanne Gabrielsson, Maria Eldh Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but sti (show more...)Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget. (hide) EV-METRIC 71% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Blood plasma Focus vesicles extracellular 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 Density gradient Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes EV density (g/ml) 1.12-1.19 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MM6 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 130000 Duration (min) 960 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 65 Pelleting: duration (min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method Bradford Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database No Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 126 Particle analysis: flow cytometry Flow cytometer type BD FACS Canto II Hardware adjustment Calibration bead size 4 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210020	5/9	Homo sapiens	MM6	(d)(U)C Filtration DG	Veerman, Rosanne	2021	71%
Study summary Full title Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin All authors Rosanne E. Veerman, Loes Teeuwen, Paulo Czarnewski, Gözde Güclüler Akpinar, AnnSofi Sandberg, Xiaofang Cao, Maria Pernemalm, Lukas M. Orre, Susanne Gabrielsson, Maria Eldh Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but sti (show more...)Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget. (hide) EV-METRIC 71% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Cell culture supernatant Focus vesicles extracellular 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 Density gradient Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes EV density (g/ml) 1.12-1.19 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MM6 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 1440 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 65 Pelleting: duration (min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method Bradford Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database No Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 92 Particle analysis: flow cytometry Flow cytometer type BD FACS Canto II Hardware adjustment Calibration bead size 4 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200102	7/7	Homo sapiens	Blood plasma	DG	Tóth, Eszter	2021	71%
Study summary Full title Formation of a protein corona on the surface of extracellular vesicles in blood plasma All authors Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás Journal J Extracell Vesicles Abstract In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide) EV-METRIC 71% (95th 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 Rheuma: EV-depleted plasma, spiked with THP1 EVs Focus vesicles extracellular 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 Density gradient Protein markers EV: CD63/ Phosphatydilserine non-EV: None Proteomics yes EV density (g/ml) 1.10-1.15 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: duration (min) 80 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry FACS Calibur Calibration bead size The vesicular gate was set using Megamix Beads (Bi Antibody details provided? No Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type FACS Calibur Hardware adjustment Calibration bead size 0.160;0.200;0.240;0.500 Report type Not Reported

	EV220194	3/6	Mus musculus	Liver tissue	(d)(U)C Filtration	Matejovič A	2021	67%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 67% (83rd 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 Liver tissue Sample origin Cut liver - CCL4-induced hepatotoxicity 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: calnexin/ RPL5 Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Liver 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 Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 210053 Filtration steps 0.2 or 0.22 µm Other Name other separation method Filtration Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ HSP70/ TSG101 Not detected contaminants calnexin/ RPL5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 117 ± 40.6 nm and 287 ± 65.9 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: enzymatic digestion (2 mg/mL collagenase D and 40 U/mL DNase I) and 30 min incubation at 37 Celsius degrees/ tissue cutting (1x1 mm pieces)

	EV220194	4/6	Mus musculus	Skeletal muscle tissue	(d)(U)C Filtration	Matejovič A	2021	67%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 67% (50th 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 Skeletal muscle tissue Sample origin Cut skeletal muscle - cardiotoxin-induced injury 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: RPL5/ calnexin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Skeletal muscle 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 Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 210053 Filtration steps 0.2 or 0.22 µm Other Name other separation method Filtration Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ HSP70/ TSG101 Detected contaminants calnexin Not detected contaminants RPL5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) a proportion of large EVs (260 ± 80.8 nm) among the small EVs (88 ± 35.5 nm) EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: enzymatic digestion (2 mg/mL collagenase D and 40 U/mL DNase I) and 30 min incubation at 37 Celsius degrees/ tissue cutting (1x1 mm pieces)

	EV220194	5/6	Mus musculus	Heart tissue	(d)(U)C Filtration	Matejovič A	2021	67%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 67% (85th 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 Heart tissue Sample origin Cut heart - doxorubicin-induced cardiomyopathy 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 (Differential) (ultra)centrifugation Filtration Protein markers EV: Alix/ HSP70/ TSG101/ CD63 non-EV: calnexin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Heart 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 Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 210053 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ HSP70/ TSG101 Not detected EV-associated proteins CD63 Detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 108 ± 33.5 nm and 398 ± 167.5 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: enzymatic digestion (2 mg/mL collagenase D and 40 U/mL DNase I) and 30 min incubation at 37 Celsius degrees/ tissue cutting (1x1 mm pieces)

	EV220194	6/6	Mus musculus	Heart tissue	(d)(U)C DC Filtration	Matejovič A	2021	67%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 67% (85th 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 Heart tissue Sample origin Cut heart - doxorubicin-induced cardiomyopathy 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 (Differential) (ultra)centrifugation Density cushion Filtration Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: calnexin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Heart 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 Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 210053 Wash: volume per pellet (ml) 11 mL (PBS) Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 210053 Filtration steps 0.2 or 0.22 µm Density cushion Density medium Sucrose Sample volume 11 Cushion volume 1 Density of the cushion 30% Centrifugation time 60 Centrifugation speed 210053 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ HSP70/ TSG101 Detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 125 ± 39.8 nm and 398 ± 167.5 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: enzymatic digestion (2 mg/mL collagenase D and 40 U/mL DNase I) and 30 min incubation at 37 Celsius degrees/ tissue cutting (1x1 mm pieces)

	EV210261	3/8	Homo sapiens	LIM1863	(d)(U)C DG	Rai, Alin	2021	67%
Study summary Full title Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform All authors Alin Rai, Haoyun Fang, Bethany Claridge, Richard J. Simpson, and David W Greening Journal J Extracell Vesicles Abstract The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gatewa (show more...)The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function. (hide) EV-METRIC 67% (94th 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 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 Protein markers EV: CD63 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LIM1863 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database Yes Characterization: Lipid analysis No

	EV210261	4/8	Homo sapiens	LIM1863	(d)(U)C DG	Rai, Alin	2021	67%
Study summary Full title Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform All authors Alin Rai, Haoyun Fang, Bethany Claridge, Richard J. Simpson, and David W Greening Journal J Extracell Vesicles Abstract The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gatewa (show more...)The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function. (hide) EV-METRIC 67% (94th 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 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 Protein markers EV: CD63 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LIM1863 EV-harvesting Medium Serum free medium 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) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database Yes Characterization: Lipid analysis No

	EV210261	5/8	Homo sapiens	MDA MB 231	(d)(U)C DG	Rai, Alin	2021	67%
Study summary Full title Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform All authors Alin Rai, Haoyun Fang, Bethany Claridge, Richard J. Simpson, and David W Greening Journal J Extracell Vesicles Abstract The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gatewa (show more...)The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function. (hide) EV-METRIC 67% (94th 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 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 Protein markers EV: CD63 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA MB 231 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database Yes Characterization: Lipid analysis No

	EV210261	6/8	Homo sapiens	MDA MB 231	(d)(U)C DG	Rai, Alin	2021	67%
Study summary Full title Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform All authors Alin Rai, Haoyun Fang, Bethany Claridge, Richard J. Simpson, and David W Greening Journal J Extracell Vesicles Abstract The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gatewa (show more...)The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function. (hide) EV-METRIC 67% (94th 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 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 Protein markers EV: CD63 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA MB 231 EV-harvesting Medium Serum free medium 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) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database Yes Characterization: Lipid analysis No

	EV210261	7/8	Homo sapiens	U87	(d)(U)C DG	Rai, Alin	2021	67%
Study summary Full title Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform All authors Alin Rai, Haoyun Fang, Bethany Claridge, Richard J. Simpson, and David W Greening Journal J Extracell Vesicles Abstract The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gatewa (show more...)The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function. (hide) EV-METRIC 67% (94th 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 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 Protein markers EV: CD63 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database Yes Characterization: Lipid analysis No

	EV210261	8/8	Homo sapiens	U87	(d)(U)C DG	Rai, Alin	2021	67%
Study summary Full title Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform All authors Alin Rai, Haoyun Fang, Bethany Claridge, Richard J. Simpson, and David W Greening Journal J Extracell Vesicles Abstract The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gatewa (show more...)The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function. (hide) EV-METRIC 67% (94th 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 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 Protein markers EV: CD63 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.11 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 EV-harvesting Medium Serum free medium 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) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Proteomics database Yes Characterization: Lipid analysis No

	EV210253	1/1	Homo sapiens	Serum	(d)(U)C	Małys MSS	2021	67%
Study summary Full title Isolation of Small Extracellular Vesicles from Human Sera. All authors Małys MSS, Aigner C, Schulz SMM, Schachner H, Rees AJJ, Kain R Journal Int J Mol Sci Abstract Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are n (show more...)Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exo-spin™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations. (hide) EV-METRIC 67% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles small 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 (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ MHC1/ MHC2/ CD19/ CD3/ CD4 non-EV: Calnexin/ Albumin/ apoB Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods/ Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum 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: rotor type SX4750 in Allegra X-15R Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 32 Wash: time (min) 120 Wash: Rotor Type SX4750 in Allegra X-15R Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 32.4 ?g Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63 Detected contaminants Calnexin Not detected contaminants Albumin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Antibody details provided? Yes Antibody dilution provided? No Selected surface protein(s) CD9/ CD63/ CD81/ MHC1/ MHC2/ CD19/ CD3/ CD4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes EM EM-type Transmission EM/ Immuno EM EM protein CD63/ CD9/ ApoB100/48 Image type Close-up

	EV210253	2/1	Homo sapiens	Serum	(d)(U)C Exo-spin	Małys MSS	2021	67%
Study summary Full title Isolation of Small Extracellular Vesicles from Human Sera. All authors Małys MSS, Aigner C, Schulz SMM, Schachner H, Rees AJJ, Kain R Journal Int J Mol Sci Abstract Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are n (show more...)Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exo-spin™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations. (hide) EV-METRIC 67% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles small 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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD9/ CD63/ CD81/ MHC1/ MHC2/ CD19/ CD3/ CD4 non-EV: Calnexin/ Albumin/ apoB Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods/ Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit Exo-spin Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 40 ?g Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63 Detected contaminants Calnexin Not detected contaminants Albumin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Antibody details provided? Yes Antibody dilution provided? No Selected surface protein(s) CD9/ CD63/ CD81/ MHC1/ MHC2/ CD19/ CD3/ CD4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 132 EV concentration Yes EM EM-type Transmission EM/ Immuno EM EM protein CD63/ CD9/ ApoB100/48 Image type Close-up

	EV210179	1/6	Mus musculus	Renca	(d)(U)C Exo-Spin	Samoylenko, Anatoliy	2021	67%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (hide) EV-METRIC 67% (94th 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 extracellular 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 (d)(U)C Commercial method Protein markers EV: CD81/ TSG101/ CD9/ Alix non-EV: Argonaute2/ GM130 Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Renca EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count Not reported 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) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Commercial kit Exo-Spin Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per million cells 1.15 Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101/ Alix/ CD81 Not detected contaminants GM130/ Argonaute2 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131 EV concentration Yes Particle yield Yes, as number of particles per million cells 9.07E+07 EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Wide-field Report size (nm) 30-200

	EV210179	3/6	Mus musculus	Renca	(d)(U)C Exo-Spin	Samoylenko, Anatoliy	2021	67%
Study summary Full title Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles All authors Anatoliy Samoylenko, Martin Kögler, Artem Zhyvolozhnyi, Olha Makieieva, Geneviève Bart, Sampson S. Andoh, Matthieu Roussey, Seppo J. Vainio, and Jussi Hiltunen Journal Sci Rep Abstract Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles invo (show more...)Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols. (hide) EV-METRIC 67% (94th 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 hypoxia Focus vesicles extracellular 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 (d)(U)C Commercial method Protein markers EV: CD81/ TSG101/ CD9/ Alix non-EV: Argonaute2/ GM130 Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Renca EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count Not reported 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) 900 Pelleting: rotor type TH-641 Pelleting: speed (g) 100000 Commercial kit Exo-Spin Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) Yes, per cell 1.53 Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ TSG101/ CD81 Not detected contaminants GM130/ Argonaute2 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 168 EV concentration Yes Particle yield Yes, as number of particles per million cells 1.73E+08 EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Wide-field Report size (nm) 30-200

	EV210167	1/4	Homo sapiens	A375SM	(d)(U)C	Torii, Chisaho	2021	67%
Study summary Full title miRNA-1246 in extracellular vesicles secreted from metastatic tumor induces drug resistance in tumor endothelial cells All authors Chisaho Torii, Nako Maishi, Taisuke Kawamoto, Masahiro Morimoto, Kosuke Akiyama, Yusuke Yoshioka, Takashi Minami, Takuya Tsumita, Mohammad Towfik Alam, Takahiro Ochiya, Yasuhiro Hida, Kyoko Hida Journal Sci Rep Abstract Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs (show more...)Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of P-glycoprotein (P-gp, ABCB1), contrary to traditional assumptions. Furthermore, P-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential P-gp expression in TECs remains unclear. miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on EVs and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs. (hide) EV-METRIC 67% (94th 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 extracellular 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 (d)(U)C Protein markers EV: HSP70/ CD63/ CD9 non-EV: Cytochrome C Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A375SM EV-harvesting Medium EV-depleted medium Preparation of EDS 70 minutes at >=100,000g Cell count 3.00E+06 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) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 60 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ HSP70 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 140 EM EM-type Transmission-EM Image type Close-up

	EV210167	2/4	Homo sapiens	A375	(d)(U)C	Torii, Chisaho	2021	67%
Study summary Full title miRNA-1246 in extracellular vesicles secreted from metastatic tumor induces drug resistance in tumor endothelial cells All authors Chisaho Torii, Nako Maishi, Taisuke Kawamoto, Masahiro Morimoto, Kosuke Akiyama, Yusuke Yoshioka, Takashi Minami, Takuya Tsumita, Mohammad Towfik Alam, Takahiro Ochiya, Yasuhiro Hida, Kyoko Hida Journal Sci Rep Abstract Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs (show more...)Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of P-glycoprotein (P-gp, ABCB1), contrary to traditional assumptions. Furthermore, P-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential P-gp expression in TECs remains unclear. miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on EVs and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs. (hide) EV-METRIC 67% (94th 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 extracellular 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 (d)(U)C Protein markers EV: HSP70/ CD63/ CD9 non-EV: Cytochrome C Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A375 EV-harvesting Medium EV-depleted medium Preparation of EDS 70 minutes at >=100,000g Cell count 3.00E+06 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) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 60 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ HSP70 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 140 EM EM-type Transmission-EM Image type Close-up

	EV210163	1/5	Homo sapiens	Decidual	(d)(U)C SEC (non-commercial) UF Filtration	Shepherd Megan	2021	67%
Study summary Full title Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response All authors Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon Journal Cell Commun Signal Abstract Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide) EV-METRIC 67% (94th 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 Size-exclusion chromatography (non-commercial) Ultrafiltration Filtration Protein markers EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9 non-EV: GM130 Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Decidual EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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: rotor type Type 70.1Ti Pelleting: speed (g) 100 000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0,1 Sample volume/column (mL) 0,1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ CD9/ CD63/ ANIXA S/ iCAM/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD81/ Flotillin1/ ANIXA S/ iCAM/ TSG101/ CD63/ CD9/ Alix Detected contaminants GM130 Not detected contaminants GM130 Proteomics database No Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 115 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.00e+0 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up Report size (nm) 100

	EV210163	2/5	Homo sapiens	Myometrial	(d)(U)C SEC (non-commercial) UF Filtration	Shepherd Megan	2021	67%
Study summary Full title Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response All authors Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon Journal Cell Commun Signal Abstract Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide) EV-METRIC 67% (94th 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 Size-exclusion chromatography (non-commercial) Ultrafiltration Filtration Protein markers EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9 non-EV: GM130 Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Myometrial EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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: rotor type Type 70.1Ti Pelleting: speed (g) 100 000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0,1 Sample volume/column (mL) 0,1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ CD9/ CD63/ ANIXA S/ iCAM/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD81/ Flotillin1/ ANIXA S/ iCAM/ TSG101/ CD63/ CD9/ Alix Detected contaminants GM130 Proteomics database No Detected EV-associated proteins CD81/ CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 116,075 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.00e+0 EM EM-type Cryo-EM Image type Close-up Report size (nm) 100

	EV210163	3/5	Homo sapiens	Decidual	(d)(U)C SEC (non-commercial) UF Filtration	Shepherd Megan	2021	67%
Study summary Full title Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response All authors Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon Journal Cell Commun Signal Abstract Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide) EV-METRIC 67% (94th 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 Oxidative stress inducer (CSE) treated 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 Size-exclusion chromatography (non-commercial) Ultrafiltration Filtration Protein markers EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9 non-EV: GM130 Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Decidual EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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: rotor type Type 70.1Ti Pelleting: speed (g) 100 000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0,1 Sample volume/column (mL) 0,1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ CD9/ CD63/ TSG101/ ANIXA S/ iCAM/ CD81 Detected contaminants GM130 Proteomics database No Detected EV-associated proteins CD81/ CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 116 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.00e+0 EM EM-type Cryo-EM Image type Close-up Report size (nm) 100

	EV210163	4/5	Homo sapiens	Myometrial	(d)(U)C SEC (non-commercial) UF Filtration	Shepherd Megan	2021	67%
Study summary Full title Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response All authors Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon Journal Cell Commun Signal Abstract Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide) EV-METRIC 67% (94th 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 Oxidative stress inducer (CSE) treated 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 Size-exclusion chromatography (non-commercial) Ultrafiltration Filtration Protein markers EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9 non-EV: GM130 Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Myometrial EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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: rotor type Type 70.1Ti Pelleting: speed (g) 100 000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0,1 Sample volume/column (mL) 0,1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ ANIXA S/ iCAM/ CD9/ CD63/ TSG101/ CD81 Detected contaminants GM130 Proteomics database No Detected EV-associated proteins CD81/ CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 116 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.00e+0 EM EM-type Cryo-EM Image type Close-up Report size (nm) 90

	EV210163	5/5	Homo sapiens	Myometrial	(d)(U)C SEC (non-commercial) UF Filtration	Shepherd Megan	2021	67%
Study summary Full title Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response All authors Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon Journal Cell Commun Signal Abstract Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide) EV-METRIC 67% (94th 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 Inflammation inducer-TNF-alpha treated 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 Size-exclusion chromatography (non-commercial) Ultrafiltration Filtration Protein markers EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9 non-EV: GM130 Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Myometrial EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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: rotor type Type 70.1Ti Pelleting: speed (g) 100 000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0,1 Sample volume/column (mL) 0,1 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ ANIXA S/ iCAM/ CD9/ CD63/ TSG101/ Alix/ CD81 Detected contaminants GM130 Proteomics database No Detected EV-associated proteins CD81/ CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 120 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.00e+0 EM EM-type Cryo-EM Image type Close-up Report size (nm) 90

	EV210158	1/2	Bos taurus	Mac-T cells	(d)(U)C Filtration	Ogunnaike, Mojisola	2021	67%
Study summary Full title Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery All authors Mojisola Ogunnaike, Haichuan Wang, Janos Zempleni Journal Int J Pharma Abstract Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BM (show more...)Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BMEs originate in mammary alveolar cells. Here, we determined whether bovine mammary alveolar MAC-T cells afford a tool to assess RNA delivery by BMEs. MAC-T cells exosomes (MAC-T BMEs) and BMEs were harvested by differential ultracentrifugation. Exosome size, morphology, microRNA content and marker proteins were assessed using nanoparticle tracking analysis, transmission electron microscopy, real-time PCR and immunoblot analysis, respectively. MAC-T cells were genetically engineered to secrete MAC-T BMEs endogenously labeled with a near-infrared fluorescent protein and tissue distribution was compared to fluorophore-labeled BMEs following intravenous injection in C57BL/6 mice. Morphology and size were similar in MAC-T BMEs and BMEs (94 ± 5.8 nm and 101 ± 4.2 nm, p > 0.05). Both preparations expressed miR-320a, miR-200c and let-7a-5p (positive controls) but not miR-1 (negative control). Exosome marker proteins, CD9, CD63, CD81 and Tsg101, were detected in both MAC-T BMEs and BMEs. Distribution in mouse tissues was similar for both preparations, with liver being the primary accumulation site. Collectively, MAC-T BMEs afford a tool for BMEs-based RNA delivery studies. (hide) EV-METRIC 67% (94th 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 (d)(U)C Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9 non-EV: Calnexin/ Histone H3 Proteomics no Show all info Study aim Exosomes and cargo characterization Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells Mac-T cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 97 Cell count 6,20E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type F37L-8100 rotor Pelleting: speed (g) 130000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected contaminants Histone H3/ Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 100 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 94 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6,11E+08 EM EM-type Transmission-EM Image type Wide-field Report size (nm) ~90

	EV210158	2/2	Bos taurus	Bovine milk	(d)(U)C Filtration	Ogunnaike, Mojisola	2021	67%
Study summary Full title Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery All authors Mojisola Ogunnaike, Haichuan Wang, Janos Zempleni Journal Int J Pharma Abstract Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BM (show more...)Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BMEs originate in mammary alveolar cells. Here, we determined whether bovine mammary alveolar MAC-T cells afford a tool to assess RNA delivery by BMEs. MAC-T cells exosomes (MAC-T BMEs) and BMEs were harvested by differential ultracentrifugation. Exosome size, morphology, microRNA content and marker proteins were assessed using nanoparticle tracking analysis, transmission electron microscopy, real-time PCR and immunoblot analysis, respectively. MAC-T cells were genetically engineered to secrete MAC-T BMEs endogenously labeled with a near-infrared fluorescent protein and tissue distribution was compared to fluorophore-labeled BMEs following intravenous injection in C57BL/6 mice. Morphology and size were similar in MAC-T BMEs and BMEs (94 ± 5.8 nm and 101 ± 4.2 nm, p > 0.05). Both preparations expressed miR-320a, miR-200c and let-7a-5p (positive controls) but not miR-1 (negative control). Exosome marker proteins, CD9, CD63, CD81 and Tsg101, were detected in both MAC-T BMEs and BMEs. Distribution in mouse tissues was similar for both preparations, with liver being the primary accumulation site. Collectively, MAC-T BMEs afford a tool for BMEs-based RNA delivery studies. (hide) EV-METRIC 67% (87th 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 Bovine milk 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 (d)(U)C Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9 non-EV: Calnexin/ Histone H3 Proteomics no Show all info Study aim Exosomes and cargo characterization Sample Species Bos taurus Sample Type Bovine milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type F37L-8100 rotor Pelleting: speed (g) 130000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected contaminants Histone H3/ Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 100 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 101 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3,40E+10 EM EM-type Transmission-EM Image type Wide-field Report size (nm) ~100

	EV210127	1/9	Homo sapiens	SK-MEL-147	(d)(U)C	García-Silva, Susana	2021	67%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small 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 Protein markers EV: Alix/ RAB27a/ NGFR/ GAPDH non-EV: Calnexin/ GM130 Proteomics yes EV density (g/ml) Not specified Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS 70 min at 100,000g;Other preparation Cell viability (%) 90 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 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 7.7 Sample volume (mL) 0.2 Orientation Top-down Rotor type Type 70 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.75 Fraction processing Ultracentrifugation Pelleting: volume per fraction 3.25 Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins NGFR/ GAPDH/ Alix/ CD81/ CD63 Not detected EV-associated proteins RAB27a Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Antibody details provided? No Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 130 EV concentration Yes Particle yield Yes, as number of particles per million cells 5,70E+09

	EV210127	5/9	Mus musculus	B16-F1	(d)(U)C	García-Silva, Susana	2021	67%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small 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 Protein markers EV: Alix/ NGFR non-EV: Calnexin/ GM130 Proteomics yes EV density (g/ml) Not specified Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16-F1 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;70 min at 100,000g Cell viability (%) 92 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 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 7.7 Sample volume (mL) 0.2 Orientation Top-down Rotor type Type 70 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.75 Fraction processing Ultracentrifugation Pelleting: volume per fraction 3.25 Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins NGFR/ Alix/ CD81/ CD9 Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Antibody details provided? No Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145 EV concentration Yes Particle yield Yes, as number of particles per million cells 6,00E+09

	EV210127	9/9	Mus musculus	B16-F10	(d)(U)C	García-Silva, Susana	2021	67%
Study summary Full title Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through a NGFR-dependent mechanism All authors Susana García-Silva, Alberto Benito-Martín, Laura Nogués, Alberto Hernández-Barranco, Marina S. Mazariegos, Vanesa Santos, Marta Hergueta-Redondo, Pilar Ximénez-Embún, Raghu P. Kataru, Ana Amor Lopez, Cristina Merino, Sara Sánchez-Redondo, Osvaldo Graña-Castro, Irina Matei, José Ángel Nicolás-Avila, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Lola Martínez, Manuel Pérez-Martínez, Gadea Mata, Anna Szumera-Ciećkiewicz, Iwona Kalinowska, Annalisa Saltari, Julia M. Martínez-Gómez, Sabrina A. Hogan, H. Uri Saragovi, Sagrario Ortega, Carmen Garcia-Martin, Jasminka Boskovic, Mitchell P. Levesque, Piotr Rutkowski, Andrés Hidalgo, Javier Muñoz, Diego Megías, Babak J. Mehrara, David Lyden and Héctor Peinado. Journal Nat. Cancer Abstract Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metast (show more...)Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we have analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK, NF-kB activation and ICAM-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR/p75NTR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to matched primary tumors and frequency of NGFR+ metastatic melanoma cells in lymph node correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs reinforcing lymph node pre-metastatic niche formation and metastasis. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small 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 Protein markers EV: Alix/ NGFR non-EV: Calnexin/ GM130 Proteomics yes EV density (g/ml) Not specified Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16-F10 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;70 min at 100,000g Cell viability (%) 94 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 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 7.7 Sample volume (mL) 0.2 Orientation Top-down Rotor type Type 70 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.75 Fraction processing Ultracentrifugation Pelleting: volume per fraction 3.25 Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ NGFR/ CD81/ CD9 Not detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry FACS Canto Calibration bead size 0,2 Antibody details provided? No Detected EV-associated proteins NGFR Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 149 EV concentration Yes Particle yield Yes, as number of particles per million cells 3000000000

	EV210121	1/9	Homo sapiens	Immortalized ectocervical epithelial cell	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized ectocervical epithelial cell EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 5.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 110 EV concentration Yes Particle yield particles per cell;Yes, other: 21 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 110

	EV210121	2/9	Homo sapiens	Immortalized ectocervical epithelial cell	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 LPS treatment 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized ectocervical epithelial cell EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 5.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 106 EV concentration Yes Particle yield particles per cell;Yes, other: 51 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 111

	EV210121	3/9	Homo sapiens	Immortalized ectocervical epithelial cell	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 Cigarette smoke extract treatment 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized ectocervical epithelial cell EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 5.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108 EV concentration Yes Particle yield particles per cell;Yes, other: 280 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 106

	EV210121	4/9	Homo sapiens	Immortalized endocervical epithelial cell	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized endocervical epithelial cell EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 3.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Detected EV-associated proteins CD81/ CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 110 EV concentration Yes Particle yield particles per cell;Yes, other: 73 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 112

	EV210121	5/9	Homo sapiens	Immortalized endocervical epithelial cell	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 LPS treatment 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized endocervical epithelial cell EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 3.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 115 EV concentration Yes Particle yield particles per cell;Yes, other: 63 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 115

	EV210121	6/9	Homo sapiens	Immortalized endocervical epithelial cell	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 Cigarette smoke extract treatment 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized endocervical epithelial cell EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 3.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 109 EV concentration Yes Particle yield particles per cell;Yes, other: 226 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120

	EV210121	7/9	Homo sapiens	Immortalized cervical stromal cells	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized cervical stromal cells EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Detected EV-associated proteins CD81/ CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 104 EV concentration Yes Particle yield particles per cell;Yes, other: 35 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 95

	EV210121	8/9	Homo sapiens	Immortalized cervical stromal cells	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 LPS treatment 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized cervical stromal cells EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 116 EV concentration Yes Particle yield particles per cell;Yes, other: 124 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 105

	EV210121	9/9	Homo sapiens	Immortalized cervical stromal cells	(d)(U)C SEC (non-commercial) Filtration	Tantengco, Ourlad Alzeus	2021	67%
Study summary Full title Cross talk: Trafficking and functional impact of maternal exosomes at the Feto-maternal Interface under normal and pathologic states All authors Ourlad Alzeus G Tantengco, Enkhtuya Radnaa, Hend Shahin, Talar Kechichian, Ramkumar Menon Journal Biology of Reproduction Abstract Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and (show more...)Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (Ecto), endocervical epithelial cells (Endo), and cervical stromal cells (Stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual, chorion trophoblast (CTC), chorion mesenchymal (CMC), amnion mesenchymal (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. ELISA for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi. (hide) EV-METRIC 67% (94th 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 Cigarette smoke extract treatment 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 (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD63 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Immortalized cervical stromal cells EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 0.5 Sample volume/column (mL) 0.1 Resin type Not Specified Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 118 EV concentration Yes Particle yield particles per cell;Yes, other: 98 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 115

	EV210120	1/1	Homo sapiens	bone marrow-derived mesenchymal stem cells	PEG precipitation (d)(U)C	Cone, Allaura	2021	67%
Study summary Full title Mesenchymal stem cell-derived extracellular vesicles ameliorate Alzheimer's disease-like phenotypes in a preclinical mouse model All authors Allaura S Cone, Xuegang Yuan, Li Sun, Leanne C Duke, Michael P Vreones, Allison N Carrier, Stephanie M Kenyon, Spencer R Carver, Sarah D Benthem, Alina C Stimmell, Shawn C Moseley, David Hike, Samuel C Grant, Aaron A Wilber, James M Olcese, David G Meckes Jr Journal Theranostics Abstract Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects more than 44 mil (show more...)Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects more than 44 million people worldwide. Despite the high disease burden, there is no effective treatment for people suffering from AD. Mesenchymal stem cells (MSCs) are multipotent stromal cells that have been widely studied due to their therapeutic potential. However, administration of cells has been found to have a multitude of limitations. Recently, extracellular vesicles (EVs) derived from MSCs have been studied as a therapeutic candidate, as they exhibit similar immunoprotective and immunomodulatory abilities as the host human MSCs. Methods: To test the potential therapeutic effects of MSC EVs, human bone-marrow derived MSCs were grown in three-dimensional (3D) cell culture, and small EVs were harvested using differential ultracentrifugation. These small EVs were given to non-transgenic (NT) or 5XFAD (5 familial Alzheimer's disease mutations) mice intranasally (IN) every 4 days for 4 months. The mice were then required to perform a variety of behavioral assays to measure changes in learning and memory. Afterwards, immunohistochemistry was performed on brain slices to measure amyloid beta (Aβ) and glial fibrillary acidic protein (GFAP) levels. Results: The data revealed that 5XFAD mice that received hMSC-EV treatment behaved significantly better in cognitive tests than saline treated 5XFAD mice, with no significant change between EV-treated 5XFAD mice and NT mice. Additionally, we found lower Aβ plaque load in the hippocampus of the EV-treated mice. Finally, less colocalization between GFAP and Aβ plaques was found in the brain of EV-treated mice compared to saline. Conclusions: Taken together, these data suggest that IN administration of MSC-derived EVs can slow down AD pathogenesis. (hide) EV-METRIC 67% (94th 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 extracellular 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 PEG precipitation (d)(U)C Protein markers EV: TSG101/ CD63/ CD9/ Syntenin-1 non-EV: Calnexin Proteomics no Show all info Study aim Therapeutic Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells bone marrow-derived mesenchymal stem cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1E5-2E5 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) 60 Pelleting: rotor type S-4-104 Pelleting: speed (g) 3200 Wash: volume per pellet (ml) 1 Wash: time (min) 70 Wash: Rotor Type TLA-120.2 Wash: speed (g) 120000 Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method None Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Syntenin-1/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.00E+10 EM EM-type Transmission-EM Image type Wide-field

	EV210109	1/3	Homo sapiens	Glioma cells	(d)(U)C	Cai, Tanxi	2021	67%
Study summary Full title LncRNA-encoded microproteins: A new form of cargo in cell culture-derived and circulating extracellular vesicles All authors Tanxi Cai, Qing Zhang, Bowen Wu, Jifeng Wang, Na Li, Tingting Zhang, Zhipeng Wang, Jianjun Luo, Xiaojing Guo, Xiang Ding, Zhensheng Xie, Lili Niu, Weihai Ning, Zhen Fan, Xiaowei Chen, Xiangqian Guo, Runsheng Chen, Hongwei Zhang, Fuquan Yang Journal J Extracell Vesicles Abstract Advancements in omics-based technologies over the past few years have led to the discovery of numero (show more...)Advancements in omics-based technologies over the past few years have led to the discovery of numerous biologically relevant peptides encoded by small open reading frames (smORFs) embedded in long noncoding RNA (lncRNA) transcripts (referred to as microproteins here) in a variety of species. However, the mechanisms and modes of action that underlie the roles of microproteins have yet to be fully characterized. Herein, we provide the first experimental evidence of abundant microproteins in extracellular vesicles (EVs) derived from glioma cancer cells, indicating that the EV-mediated transfer of microproteins may represent a novel mechanism for intercellular communication. Intriguingly, when examining human plasma, 48, 11 and 3 microproteins were identified from purified EVs, whole plasma and EV-free plasma, respectively, suggesting that circulating microproteins are primarily enriched in EVs. Most importantly, the preliminary data showed that the expression profile of EV microproteins in glioma patient diverged from the health donors, suggesting that the circulating microproteins in EVs might have potential diagnostic application in identifying patients with glioma. (hide) EV-METRIC 67% (94th 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 extracellular 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 (d)(U)C Protein markers EV: Alix/ CD81/ CD63/ CD9/ Annexin A1 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioma cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Cell count 4.00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 70 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Annexin A1/ Alix/ CD81 Not detected contaminants Calnexin Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 124.9+/-7.9 EV concentration Yes Particle yield Yes, as number of particles per million cells 4.00E+08 EM EM-type Transmission-EM Image type Wide-field

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