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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210126	4/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 78% (97th 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 CD63-GFP 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: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD12 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 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) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ GFP/ CD63/ TSG101/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 74 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	5/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 78% (97th 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 CD81TM-GFP 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: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD13 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 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) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 74 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	6/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 78% (97th 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 SDCBP-GFP 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: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD14 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 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) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 76 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	7/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 78% (97th 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 APMAP-GFP 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: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD15 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 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) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 76 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	8/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 78% (97th 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 CD63TM-GFP 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: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD16 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 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) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 73 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210105	1/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	78%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 78% (97th 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 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: CD63/ Basigin/ SLC3A2/ Syntenin/ CD9/ CD81 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 6 Wash: time (min) 70 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ Syntenin/ Basigin/ SLC3A2/ CD81 Detected contaminants acetylcholinesterase Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-400 EV concentration Yes Particle yield No NA EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field Report size (nm) 40-300

	EV210105	7/14	Homo sapiens	HeLa	(d)(U)C	Mathieu, Mathilde	2021	78%
Study summary Full title Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9 All authors Mathilde Mathieu, Nathalie Névo, Mabel Jouve, José Ignacio Valenzuela, Mathieu Maurin, Frederik Verweij, Roberta Palmulli, Danielle Lankar, Florent Dingli, Damarys Loew,Eric Rubinstein, Gaëlle Boncompain, Franck Perez & Clotilde Théry Journal Nat Commun Abstract Despite their roles in intercellular communications, the different populations of extracellular vesi (show more...)Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type. (hide) EV-METRIC 78% (97th 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 BafA1 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 (d)(U)C Protein markers EV: CD63/ CD81/ CD9/ basigin/ syntenin/ SLC3A2 non-EV: Calnexin/ acetylcholinesterase Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HeLa EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,25E+08 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 6 Wash: time (min) 70 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD81/ syntenin/ basigin/ SLC3A2/ CD63 Detected contaminants Calnexin/ acetylcholinesterase Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-400 EV concentration Yes Particle yield No NA EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 40-300

	EV200185	1/3	Staphylococcus aureus	S. aureus NCTC6571	DG (d)(U)C Filtration	Bitto, Natalie J.	2021	78%
Study summary Full title Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy All authors Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos Journal J Extracell Vesicles Abstract Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide) EV-METRIC 78% (97th 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 / Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: S. aureus proteins non-EV: None Proteomics no EV density (g/ml) 1.069-1.119 Show all info Study aim Function Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells S. aureus NCTC6571 EV-harvesting Medium Serum free medium Cell count 2330000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P28S Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12ml Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 120 Pelleting-wash: speed (g) SW 40 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins S. aureus proteins 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 Size range/distribution Reported size (nm) 50-500 EV concentration Yes Particle yield particles per colony forming units (CFU) of bacteria;Yes, other: 1010000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200185	2/3	Staphylococcus aureus	S. aureus BPH2760	DG (d)(U)C Filtration	Bitto, Natalie J.	2021	78%
Study summary Full title Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy All authors Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos Journal J Extracell Vesicles Abstract Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide) EV-METRIC 78% (97th 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 / Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: S. aureus proteins non-EV: None Proteomics no EV density (g/ml) 1.069-1.119 Show all info Study aim Function Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells S. aureus BPH2760 EV-harvesting Medium Serum free medium Cell count 2000000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P28S Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12ml Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 120 Pelleting-wash: speed (g) SW 40 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins S. aureus proteins 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 Size range/distribution Reported size (nm) 50-500 EV concentration Yes Particle yield particles per colony forming units (CFU) of bacteria;Yes, other: 28000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200185	3/3	Staphylococcus aureus	S. aureus BPH2900	DG (d)(U)C Filtration	Bitto, Natalie J.	2021	78%
Study summary Full title Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy All authors Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos Journal J Extracell Vesicles Abstract Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (hide) EV-METRIC 78% (97th 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 / Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: S. aureus proteins non-EV: None Proteomics no EV density (g/ml) 1.069-1.119 Show all info Study aim Function Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells S. aureus BPH2900 EV-harvesting Medium Serum free medium Cell count 2130000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P28S Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12ml Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 120 Pelleting-wash: speed (g) SW 40 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins S. aureus proteins 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 Size range/distribution Reported size (nm) 50-500 EV concentration Yes Particle yield particles per colony forming units (CFU) of bacteria;Yes, other: 53000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200121	3/6	Homo sapiens	U87 glioblastoma	(d)(U)C DG	Keulers, Tom	2021	78%
Study summary Full title Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1 All authors Tom G Keulers, Sten F Libregts, Joel E J Beaumont, Kim G Savelkouls, Johan Bussink, Hans Duimel, Ludwig Dubois, Marijke I Zonneveld, Carmen López-Iglesias, Karel Bezstarosti, Jeroen A Demmers, Marc Vooijs, Marca Wauben, Kasper M A Rouschop Journal J Extracell Vesicles Abstract Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis deve (show more...)Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target. (hide) EV-METRIC 78% (97th 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 (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 glioblastoma EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1.00E+07 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 SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.4M Highest density fraction 2.0M Total gradient volume, incl. sample (mL) 12.4 Sample volume (mL) 0.15 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12.4 Pelleting: duration (min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins CD81/ CD63/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type influx Hardware adjustment customized influx Calibration bead size 100 Report type Not Reported EV concentration Yes EM EM-type Cryo-EM Image type Close-up

	EV200098	1/3	Homo sapiens	PNT1A	(d)(U)C qEV UF	Millan, Christopher	2021	78%
Study summary Full title Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D Cultures All authors Christopher Millan, Lukas Prause, Queralt Vallmajo-Martin, Natalie Hensky, Daniel Eberli Journal Advanced Healthcare Materials Abstract Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce cl (show more...)Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions. (hide) EV-METRIC 78% (97th 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 UF Protein markers EV: TSG101/ Alix/ CD9 non-EV: Calnexin/ Argonaute2 Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PNT1A EV-harvesting Medium EV-depleted medium Preparation of EDS Ultrafiltration 100kDa cutoff Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 12 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 110,000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Calnexin/ Argonaute2 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNA sequencing;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) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120

	EV200098	2/3	Homo sapiens	LNCaP	(d)(U)C qEV UF	Millan, Christopher	2021	78%
Study summary Full title Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D Cultures All authors Christopher Millan, Lukas Prause, Queralt Vallmajo-Martin, Natalie Hensky, Daniel Eberli Journal Advanced Healthcare Materials Abstract Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce cl (show more...)Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions. (hide) EV-METRIC 78% (97th 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 UF Protein markers EV: TSG101/ Alix/ GDF15/ CD9 non-EV: Calnexin/ Argonaute2 Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS Ultrafiltration 100kDa cutoff Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 12 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 110,000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Calnexin/ Argonaute2 Flow cytometry Detected EV-associated proteins CD9/ GDF15 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNAsequencing;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) 120 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Amnis ImageStreamX MkII Hardware adjustment 60x objective, 7um core diameter, low flow rate. Particles with high aspect ratio, negligible SSC, and high fluorescent were considered positively stained EVs Calibration bead size 1000 Report type Not Reported EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120

	EV200098	3/3	Homo sapiens	PC3	(d)(U)C qEV UF	Millan, Christopher	2021	78%
Study summary Full title Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D Cultures All authors Christopher Millan, Lukas Prause, Queralt Vallmajo-Martin, Natalie Hensky, Daniel Eberli Journal Advanced Healthcare Materials Abstract Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce cl (show more...)Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions. (hide) EV-METRIC 78% (97th 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 UF Protein markers EV: Alix/ TSG101/ CD9 non-EV: Calnexin/ Argonaute2 Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium EV-depleted medium Preparation of EDS Ultrafiltration 100kDa cutoff Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 12 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 110,000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected contaminants Calnexin/ Argonaute2 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNAsequencing;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) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120

	EV210281	3/16	Homo sapiens	MDAMB231	(d)(U)C UF qEV Immunoaffinity capture	Grisard, Eleonora	2021	75%
Study summary Full title Homosalate boosts the release of tumor-derived Extracellular Vesicles with anti-anoikis properties All authors Eleonora Grisard, Aurianne Lescure, Nathalie Nevo, Maxime Corbé, Mabel Jouve, Gregory Lavieu, Alain Joliot, Elaine Del Nery, Lorena Martin-Jaular, Clotilde Théry Journal bioRxiv Abstract Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular (show more...)Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63-or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs’release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we discovered that: 1) the two drugs act on EVs generated indistinct subcellular compartmentsand 2) EVs released upon treatment with Homosalate, but not with Bafilomycin A1, conferred anti-anoikis properties to another recipient tumor cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harboring distinct functional properties. (hide) EV-METRIC 75% (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 DMSO treated 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 Ultrafiltration qEV Immunoaffinity capture Protein markers EV: CD9/ CD63/ CD81/ syntenin/ CD98 non-EV: Gapdh Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Cell viability (%) 80 Cell count 27000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 kDa Membrane type PES Commercial kit qEV Immunoaffinity capture Selected surface protein(s) CD9/ CD63 Other Name other separation method qEV Other Name other separation method Immunoaffinity capture Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ syntenin/ CD98 Detected contaminants Gapdh Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100 EV concentration Yes Particle yield number of particles per million cells: 5.00e+9 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 125

	EV210281	4/16	Homo sapiens	MDAMB231	(d)(U)C UF qEV Immunoaffinity capture	Grisard, Eleonora	2021	75%
Study summary Full title Homosalate boosts the release of tumor-derived Extracellular Vesicles with anti-anoikis properties All authors Eleonora Grisard, Aurianne Lescure, Nathalie Nevo, Maxime Corbé, Mabel Jouve, Gregory Lavieu, Alain Joliot, Elaine Del Nery, Lorena Martin-Jaular, Clotilde Théry Journal bioRxiv Abstract Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular (show more...)Eukaryotic cells, including cancer cells, secrete highly heterogeneous populations of extracellular vesicles (EVs). EVs could have different subcellular origin, composition and functional properties, but tools to distinguish between EV subtypes are scarce. Here, we tagged CD63-or CD9-positive EVs secreted by triple negative breast cancer cells with Nanoluciferase enzyme, to set-up a miniaturized method to quantify secretion of these two EV subtypes directly in the supernatant of cells. We performed a cell-based high-content screening to identify clinically-approved drugs able to affect EV secretion. One of the identified hits is Homosalate, an anti-inflammatory drug found in sunscreens which robustly increased EVs’release. Comparing EVs induced by Homosalate with those induced by Bafilomycin A1, we discovered that: 1) the two drugs act on EVs generated indistinct subcellular compartmentsand 2) EVs released upon treatment with Homosalate, but not with Bafilomycin A1, conferred anti-anoikis properties to another recipient tumor cell line. In conclusion, we identified a new drug modifying EV release and demonstrated that under influence of different drugs, triple negative breast cancer cells release EV subpopulations from different subcellular origins harboring distinct functional properties. (hide) EV-METRIC 75% (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 Homosalate treated 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 Ultrafiltration qEV Immunoaffinity capture Protein markers EV: CD9/ CD63/ CD81/ syntenin/ CD98 non-EV: Gapdh Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Cell viability (%) 80 Cell count 27000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 kDa Membrane type PES Commercial kit qEV Immunoaffinity capture Selected surface protein(s) CD9/ CD63 Other Name other separation method qEV Other Name other separation method Immunoaffinity capture Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ syntenin/ CD98 Detected contaminants Gapdh Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100 EV concentration Yes Particle yield number of particles per million cells: 5.00e+9 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 125

	EV210153	1/11	Homo sapiens	22Rv1	(d)(U)C Filtration UF	Allelein, Susann	2021	75%
Study summary Full title Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations All authors Susann Allelein, Paula Medina-Perez, Ana Leonor Heitor Lopes, Sabrina Rau, Gerd Hause, Andreas Kölsch, Dirk Kuhlmeier Journal Sci Rep Abstract Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic info (show more...)Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples. (hide) EV-METRIC 75% (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 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 UF Protein markers EV: TSG101/ Alix/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 22Rv1 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.40E+11 EM EM-type Transmission electron microscopy Image type Close-up, Wide-field

	EV210153	4/11	Homo sapiens	22Rv1	(d)(U)C Filtration SEC (non-commercial)	Allelein, Susann	2021	75%
Study summary Full title Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations All authors Susann Allelein, Paula Medina-Perez, Ana Leonor Heitor Lopes, Sabrina Rau, Gerd Hause, Andreas Kölsch, Dirk Kuhlmeier Journal Sci Rep Abstract Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic info (show more...)Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples. (hide) EV-METRIC 75% (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 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 Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ Alix/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 22Rv1 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Not Specified Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.40E+11 EM EM-type Transmission electron microscopy Image type Close-up, Wide-field

	EV210153	5/11	Homo sapiens	22Rv1	(d)(U)C Filtration IAF	Allelein, Susann	2021	75%
Study summary Full title Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations All authors Susann Allelein, Paula Medina-Perez, Ana Leonor Heitor Lopes, Sabrina Rau, Gerd Hause, Andreas Kölsch, Dirk Kuhlmeier Journal Sci Rep Abstract Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic info (show more...)Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples. (hide) EV-METRIC 75% (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 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 Immunoaffinity capture (non-commercial) Protein markers EV: TSG101/ Alix/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 22Rv1 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) PSMA, CD9 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis No EM EM-type Transmission electron microscopy Image type Close-up, Wide-field

	EV210153	9/11	Homo sapiens	LNCaP	(d)(U)C Filtration UF IAF DG SEC (non-commercial)	Allelein, Susann	2021	75%
Study summary Full title Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations All authors Susann Allelein, Paula Medina-Perez, Ana Leonor Heitor Lopes, Sabrina Rau, Gerd Hause, Andreas Kölsch, Dirk Kuhlmeier Journal Sci Rep Abstract Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic info (show more...)Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples. (hide) EV-METRIC 75% (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 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 UF Immunoaffinity capture (non-commercial) DG Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ Alix/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) PSMA, CD9 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Calnexin Characterization: Lipid analysis No

	EV210144	3/9	Homo sapiens	Saliva	(d)(U)C DC	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (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 Saliva 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 DC Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 10 Cushion volume 0.7 Density of the cushion 30% Centrifugation time 120 Centrifugation speed 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63 Not detected EV-associated proteins HSC70 Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 205.7 +/- 2.3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.89E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	4/9	Homo sapiens	Saliva	(d)(U)C Chitosan-based	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (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 Saliva 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 Chitosan-based Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Other Name other separation method Chitosan-based Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63 Not detected EV-associated proteins HSC70 Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 218.2 +/- 5.4 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 8.39E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	5/9	Homo sapiens	Urine	(d)(U)C DC	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (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 Urine 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 DC Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: Tamm-Horsfall protein/ CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 10 Cushion volume 0.7 Density of the cushion 30% Centrifugation time 120 Centrifugation speed 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63 Not detected EV-associated proteins HSC70 Detected contaminants Tamm-Horsfall protein Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 165.2 +/- 0.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 7.40E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	6/9	Homo sapiens	Urine	(d)(U)C Chitosan-based	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (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 Urine 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 Chitosan-based Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: Tamm-Horsfall protein/ CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Other Name other separation method Chitosan-based Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ HSC70/ CD9/ CD63 Detected contaminants Tamm-Horsfall protein Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 136.4 +/- 5.1 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.17E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210144	7/9	Homo sapiens	HEK293	(d)(U)C DC	Kumar, Awanit	2021	75%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 75% (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 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 DC Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 7.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 10 Cushion volume 0.7 Density of the cushion 30% Centrifugation time 120 Centrifugation speed 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ HSC70/ CD9/ CD63 Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 164.8 +/- 1.6 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.08E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210109	2/3	Homo sapiens	Blood plasma	Asymmetric flow field-flow fractionation	Cai, Tanxi	2021	75%
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 75% (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 Blood plasma 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 Asymmetric flow field-flow fractionation Protein markers EV: TSG101/ CD81/ HSP90/ CD63/ CD9 non-EV: APOB/ APOA1 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Asymmetric flow field-flow fractionation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ HSP90/ TSG101/ CD81 Not detected contaminants APOA1/ APOB Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 144.2+/-2.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.00E+10 EM EM-type Transmission-EM Image type Wide-field

	EV210109	3/3	Homo sapiens	Blood plasma	Asymmetric flow field-flow fractionation	Cai, Tanxi	2021	75%
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 75% (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 Blood plasma Sample origin Giloma cancer patients 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 Asymmetric flow field-flow fractionation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Asymmetric flow field-flow fractionation Characterization: Protein analysis Protein Concentration Method BCA Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210041	1/5	Homo sapiens	SK-MEL-147	qEV	Suarez, Henar	2021	75%
Study summary Full title CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells All authors Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez‐Rivera, Soraya López‐Martín, Susana García‐Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino‐Cortez, Gabriel Barreda‐Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez‐Mó Journal J Extracell Vesicles Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundan (show more...)Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. (hide) EV-METRIC 75% (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 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: Flotillin1/ CD9/ CD63/ HSP90/ MHC1/ TSG101/ Alix/ CD81 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) >98% Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ HSP90/ MHC1/ TSG101/ Alix/ CD81 Proteomics database Yes: Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield Yes, as number of particles per million cells 1800 TRPS Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120 EV concentration Yes

	EV210041	3/5	Homo sapiens	SK-MEL-147	qEV	Suarez, Henar	2021	75%
Study summary Full title CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells All authors Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez‐Rivera, Soraya López‐Martín, Susana García‐Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino‐Cortez, Gabriel Barreda‐Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez‐Mó Journal J Extracell Vesicles Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundan (show more...)Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. (hide) EV-METRIC 75% (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 CD9 KO 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: Flotillin1/ Alix/ CD9/ CD63/ HSP90/ MHC1/ CD81 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) >98% Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Other Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD9/ CD63/ HSP90/ MHC1/ CD81 Proteomics database Yes: Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 110 EV concentration Yes Particle yield Yes, as number of particles per million cells 16000 TRPS Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 110 EV concentration Yes

	EV210041	4/5	Homo sapiens	SK-MEL-147	qEV	Suarez, Henar	2021	75%
Study summary Full title CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells All authors Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez‐Rivera, Soraya López‐Martín, Susana García‐Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino‐Cortez, Gabriel Barreda‐Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez‐Mó Journal J Extracell Vesicles Abstract Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundan (show more...)Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. (hide) EV-METRIC 75% (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 Peptide treatments 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: CD63/ Rac1/ CD81/ Flotillin1/ syntenin/ ERM/ CD9 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) >98% Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ ERM/ Rac1/ syntenin/ CD9/ CD63/ CD81 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield Yes, as number of particles per million cells 1800 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120 EV concentration Yes

	EV200183	1/2	Homo sapiens	mesenchymal stem cells derived from human Wharton's jelly	(d)(U)C Filtration Other;exoEasy Maxi Kit, Exo2D	Kim, Eun Seo	2021	75%
Study summary Full title Cellhesion VP enhances the immunomodulating potential of human mesenchymal stem cell-derived extracellular vesicles All authors Eun Seo Kim, Katsuhiko Kida, Jongsoo Mok, Yeonwoo Seong, Seo Yeon Jo, Tatsuro Kanaki, Masato Horikawa, Kyung-Hee Kim, Tae Min Kim, Tae Sub Park, Joonghoon Park Journal Biomaterials Abstract Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. Howeve (show more...)Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. However, MSCs grown under two-dimensional (2D) culture conditions differ significantly in cell shape from those in the body, with downregulated stemness genes and secretion of paracrine factors. Here, we evaluated the effect of 3D culture using Cellhesion VP, a water-insoluble material composed of chitin-based polysaccharide fibers, on the characteristics of human Wharton's jelly-derived MSCs (hMSCs). Cellhesion VP significantly increased cell proliferation after retrieval. Transcriptome analyses suggested that genes involved in cell stemness, migration ability, and extracellular vesicle (EV) production were enhanced by 3D culture. Subsequent biochemical analyses showed that the expression levels of stemness genes including OCT4, NANOG, and SSEA4 were upregulated and migration capacity was elevated in 3D-cultured hMSCs. In addition, EV production was significantly elevated in 3D cells, which contained a distinct protein profile from 2D cells. Gene and drug connectivity analyses revealed that the 2D and 3D EVs had similar functions as immunomodulators; however, 3D EVs had completely distinct therapeutic profiles for various infectious and metabolic diseases based on activation of disease-associated signaling pathways. Therefore, EVs from Cellhesion VP-primed hMSCs offer a new treatment for immune and metabolic diseases. (hide) EV-METRIC 75% (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 2D culture 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 Commercial method Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin 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 mesenchymal stem cells derived from human Wharton's jelly EV-harvesting Medium EV-depleted medium Preparation of EDS 8 hrs at 100,000 g;Other preparation Cell viability (%) 95 Cell count 6.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit Other;exoEasy Maxi Kit, Exo2D Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ TSG101/ CD81 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 171 Used for determining EV concentration? Yes NTA Report type Mean Reported size (nm) 168 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV200183	2/2	Homo sapiens	mesenchymal stem cells derived from human Wharton's jelly	(d)(U)C Filtration Other;exoEasy Maxi Kit, Exo2D	Kim, Eun Seo	2021	75%
Study summary Full title Cellhesion VP enhances the immunomodulating potential of human mesenchymal stem cell-derived extracellular vesicles All authors Eun Seo Kim, Katsuhiko Kida, Jongsoo Mok, Yeonwoo Seong, Seo Yeon Jo, Tatsuro Kanaki, Masato Horikawa, Kyung-Hee Kim, Tae Min Kim, Tae Sub Park, Joonghoon Park Journal Biomaterials Abstract Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. Howeve (show more...)Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. However, MSCs grown under two-dimensional (2D) culture conditions differ significantly in cell shape from those in the body, with downregulated stemness genes and secretion of paracrine factors. Here, we evaluated the effect of 3D culture using Cellhesion VP, a water-insoluble material composed of chitin-based polysaccharide fibers, on the characteristics of human Wharton's jelly-derived MSCs (hMSCs). Cellhesion VP significantly increased cell proliferation after retrieval. Transcriptome analyses suggested that genes involved in cell stemness, migration ability, and extracellular vesicle (EV) production were enhanced by 3D culture. Subsequent biochemical analyses showed that the expression levels of stemness genes including OCT4, NANOG, and SSEA4 were upregulated and migration capacity was elevated in 3D-cultured hMSCs. In addition, EV production was significantly elevated in 3D cells, which contained a distinct protein profile from 2D cells. Gene and drug connectivity analyses revealed that the 2D and 3D EVs had similar functions as immunomodulators; however, 3D EVs had completely distinct therapeutic profiles for various infectious and metabolic diseases based on activation of disease-associated signaling pathways. Therefore, EVs from Cellhesion VP-primed hMSCs offer a new treatment for immune and metabolic diseases. (hide) EV-METRIC 75% (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 3D culture 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 Commercial method Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin 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 mesenchymal stem cells derived from human Wharton's jelly EV-harvesting Medium EV-depleted medium Preparation of EDS 8 hrs at 100,000 g;Other preparation Cell viability (%) 95 Cell count 6.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit Other;exoEasy Maxi Kit, Exo2D Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ TSG101/ CD81 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 153 Used for determining EV concentration? Yes NTA Report type Mean Reported size (nm) 182 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV200122	1/1	Homo sapiens	293 F	Filtration	Paganini, Carolina	2021	75%
Study summary Full title Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence-Based Microfluidic Diffusion Sizing All authors Carolina Paganini, Britta Hettich, Marie R G Kopp, Adam Eördögh, Umberto Capasso Palmiero, Giorgia Adamo, Nicolas Touzet, Mauro Manno, Antonella Bongiovanni, Pablo Rivera-Fuentes, Jean-Christophe Leroux, Paolo Arosio Journal Advanced Healthcare Materials Abstract Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variet (show more...)Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time- and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development. (hide) EV-METRIC 75% (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 Filtration Protein markers EV: TSG101/ Alix/ CD63/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 293 F EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count 2.20E+08 Separation Method Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ TSG101/ Alix/ CD81 Not detected contaminants Calnexin Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 23.2+/-6.6 NTA Report type Mean Reported size (nm) 133.6+/-1.2 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.30E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200118	1/1	Homo sapiens	primary bone marrow stromal cells	(d)(U)C ExoQuick Filtration UF	Tu, Chenggong	2021	75%
Study summary Full title Endocytic pathway inhibition attenuates extracellular vesicle-induced reduction of chemosensitivity to bortezomib in multiple myeloma cells All authors Chenggong Tu, Zhimin Du, Hui Zhang, Yueyuan Feng, Yujun Qi, Yongjiang Zheng, Jinbao Liu, Jinheng Wang Journal Theranostics Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal cells (BMSCs) have been demonstrated as key factors in the progression and drug resistance of multiple myeloma (MM). EV uptake involves a variety of mechanisms which largely depend on the vesicle origin and recipient cell type. The aim of the present study was to identify the mechanisms involved in the uptake of BMSC-derived small EVs (sEVs) by MM cells, and to evaluate the anti-MM effect of targeting this process. Methods: Human BMSC-derived sEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. The effects of chemical inhibitors and shRNA-mediated knockdown of endocytosis-associated genes on sEV uptake and cell apoptosis were analyzed by flow cytometry. The anti-MM effect of blocking sEV uptake was evaluated in vitro and in a xenograft MM mouse model. Results: sEVs derived from BMSC were taken up by MM cells in a time- and dose-dependent manner, and subsequently promoted MM cell cycling and reduced their chemosensitivity to bortezomib. Chemical endocytosis inhibitors targeting heparin sulphate proteoglycans, actin, tyrosine kinase, dynamin-2, sodium/proton exchangers, or phosphoinositide 3-kinases significantly reduced MM cell internalization of BMSC-derived sEVs. Moreover, shRNA-mediated knockdown of endocytosis-associated proteins, including caveolin-1, flotillin-1, clathrin heavy chain, and dynamin-2 in MM cells suppressed sEV uptake. Furthermore, an endocytosis inhibitor targeting dynamin-2 preferentially suppressed the uptake of sEV by primary MM cells ex vivo and enhanced the anti-MM effects of bortezomib in vitro and in a mouse model. Conclusion: Clathrin- and caveolin-dependent endocytosis and macropinocytosis are the predominant routes of sEV-mediated communication between BMSCs and MM cells, and inhibiting endocytosis attenuates sEV-induced reduction of chemosensitivity to bortezomib, and thus enhances its anti-MM properties. (hide) EV-METRIC 75% (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 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 (Differential) (ultra)centrifugation Commercial method Filtration Ultrafiltration Protein markers EV: CD63/ Flotillin1/ CD9 non-EV: Calreticulin Proteomics no Show all info Study aim Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary bone marrow stromal cells EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 2000000/flask Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63 Not detected contaminants Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-210 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 9.00E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-120

	EV200102	2/7	Homo sapiens	Blood plasma	(d)(U)C	Tóth, Eszter	2021	75%
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 75% (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 Blood plasma Sample origin Control: 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 (Differential) (ultra)centrifugation Protein markers EV: CD63/ Phosphatydilserine non-EV: Albumin/ fibrinogen/ haptoglobin/ complement C3 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Wash: volume per pellet (ml) 1 Wash: time (min) 40 Wash: Rotor Type FA-45-24-11 Wash: 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 Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Detected EV-associated proteins CD63 Detected contaminants fibrinogen/ haptoglobin/ complement C3/ Albumin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 240 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 EM EM-type Immuno-EM/ Transmission-EM EM protein Other;CD63, plasma proteins Image type Close-up, Wide-field

	EV200102	5/7	Homo sapiens	Blood plasma	(d)(U)C	Tóth, Eszter	2021	75%
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 75% (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 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 (Differential) (ultra)centrifugation Protein markers EV: CD63/ Phosphatydilserine non-EV: Albumin/ fibrinogen/ haptoglobin/ complement C3 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 12500 Wash: volume per pellet (ml) 1 Wash: time (min) 40 Wash: Rotor Type FA-45-24-11 Wash: 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 Detected EV-associated proteins Phosphatydilserine Proteomics database Yes: Detected EV-associated proteins CD63 Detected contaminants fibrinogen/ haptoglobin/ complement C3/ Albumin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Not Reported 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

	EV200078	1/1	Homo sapiens	saliva	SEC (non-commercial) Filtration UF	Ogawa, Yuko	2021	75%
Study summary Full title Stability of human salivary extracellular vesicles containing dipeptidyl peptidase IV under simulated gastrointestinal tract conditions All authors Yuko Ogawa, Yoshihiro Akimoto, Mamoru Ikemoto, Yoshikuni Goto, Anna Ishikawa, Sakura Ohta, Yumi Takase, Hayato Kawakami, Masafumi Tsujimoto, Ryohei Yanoshita Journal Biochemistry and Biophysics Reports Abstract Background: Extracellular vesicles (EVs) have been isolated from various sources, including primary (show more...)Background: Extracellular vesicles (EVs) have been isolated from various sources, including primary and cultured cell lines and body fluids. Previous studies, including those conducted in our laboratory, have reported the stability of EVs under various storage conditions. Methods: EVs from human whole saliva were separated via size-exclusion chromatography. To simulate the effects of gastric or intestinal fluids on the stability of EVs, pepsin or pancreatin was added to the samples. Additionally, to determine the effect of bile acids, sodium cholate was added. The samples were then subjected to western blotting, dynamic light scattering, and transmission electron microscopy analyses. In addition, the activity of dipeptidyl peptidase (DPP) IV retained in the samples was examined to monitor the stability of EVs. Results: Under acidic conditions, with pepsin mimicking the milieu of the stomach, the EVs remained stable. However, they partially lost their membrane integrity in the presence of pancreatin and sodium cholate, indicating that they may be destabilized after passing through the duodenum. Although several associated proteins, such as mucin 5B and CD9 were degraded, DPP IV was stable, and its activity was retained under the simulated gastrointestinal conditions. Conclusion: Our data indicate that although EVs can pass through the stomach without undergoing significant damage, they may be disrupted in the intestine to release their contents. The consistent delivery of active components such as DPP IV from EVs into the intestine might play a role in the efficient modulation of homeostasis of the signal transduction pathways occurring in the gastrointestinal tract. (hide) EV-METRIC 75% (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 saliva 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 Size-exclusion chromatography (non-commercial) Filtration Ultrafiltration Protein markers EV: TSG101/ mucin 5B/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ IgA/ DPP IV/ HSP70/ CD9 non-EV: apolipoprotein B-100/ Albumin/ apolipoprotein A-1 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type saliva Separation Method Filtration steps > 0.45 µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Cellulose acetate Size-exclusion chromatography Total column volume (mL) 88 Sample volume/column (mL) 2 Resin type Sephacryl S-500 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ DPP IV/ mucin 5B/ IgA/ HSP90/ TSG101/ HSP70/ Alix/ CD81 Not detected EV-associated proteins Flotillin1 Detected contaminants apolipoprotein A-1/ apolipoprotein B-100/ Albumin Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNA sequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 40 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 40

	EV200066	1/4	Mus musculus	Primary choroid plexus epithelial cells	qEV	Vandendriessche, Charysse	2021	75%
Study summary Full title Importance of extracellular vesicle secretion at the blood–cerebrospinal fluid interface in the pathogenesis of Alzheimer’s disease All authors Charysse Vandendriessche, Sriram Balusu, Caroline Van Cauwenberghe, Marjana Brkic, Marie Pauwels, Nele Plehiers, Arnout Bruggeman, Pieter Dujardin, Griet Van Imschoot, Elien Van Wonterghem, An Hendrix, Femke Baeke, Riet De Rycke, Kris Gevaert & Roosmarijn E. Vandenbroucke Journal Acta neuropathologica communications Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in the pathog (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in the pathogenesis of Alzheimer’s disease (AD). We previously reported that the blood–cerebrospinal fluid (CSF) interface, formed by the choroid plexus epithelial (CPE) cells, releases an increased amount of EVs into the CSF in response to peripheral inflammation. Here, we studied the importance of CP-mediated EV release in AD pathogenesis. We observed increased EV levels in the CSF of young transgenic APP/PS1 mice which correlated with high amyloid beta (Aβ) CSF levels at this age. The intracerebroventricular (icv) injection of Aβ oligomers (AβO) in wild-type mice revealed a significant increase of EVs in the CSF, signifying that the presence of CSF-AβO is sufficient to induce increased EV secretion. Using in vivo, in vitro and ex vivo approaches, we identified the CP as a major source of the CSF-EVs. Interestingly, AβO-induced, CP-derived EVs induced pro-inflammatory effects in mixed cortical cultures. Proteome analysis of these EVs revealed the presence of several pro-inflammatory proteins, including the complement protein C3. Strikingly, inhibition of EV production using GW4869 resulted in protection against acute AβO-induced cognitive decline. Further research into the underlying mechanisms of this EV secretion might open up novel therapeutic strategies to impact the pathogenesis and progression of AD. (hide) EV-METRIC 75% (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 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 Commercial method No extra separation steps Protein markers EV: CD81/ CD9/ TSG101 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Primary choroid plexus epithelial cells EV-harvesting Medium Serum free medium Separation Method Commercial kit qEV Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD81/ TSG101 Not detected contaminants Calnexin Proteomics database No Detected EV-associated proteins CD9/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample F2: 9.15E09;F3: 4.42E09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200066	3/4	Mus musculus	Choroid plexus explants	qEV	Vandendriessche, Charysse	2021	75%
Study summary Full title Importance of extracellular vesicle secretion at the blood–cerebrospinal fluid interface in the pathogenesis of Alzheimer’s disease All authors Charysse Vandendriessche, Sriram Balusu, Caroline Van Cauwenberghe, Marjana Brkic, Marie Pauwels, Nele Plehiers, Arnout Bruggeman, Pieter Dujardin, Griet Van Imschoot, Elien Van Wonterghem, An Hendrix, Femke Baeke, Riet De Rycke, Kris Gevaert & Roosmarijn E. Vandenbroucke Journal Acta neuropathologica communications Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in the pathog (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in the pathogenesis of Alzheimer’s disease (AD). We previously reported that the blood–cerebrospinal fluid (CSF) interface, formed by the choroid plexus epithelial (CPE) cells, releases an increased amount of EVs into the CSF in response to peripheral inflammation. Here, we studied the importance of CP-mediated EV release in AD pathogenesis. We observed increased EV levels in the CSF of young transgenic APP/PS1 mice which correlated with high amyloid beta (Aβ) CSF levels at this age. The intracerebroventricular (icv) injection of Aβ oligomers (AβO) in wild-type mice revealed a significant increase of EVs in the CSF, signifying that the presence of CSF-AβO is sufficient to induce increased EV secretion. Using in vivo, in vitro and ex vivo approaches, we identified the CP as a major source of the CSF-EVs. Interestingly, AβO-induced, CP-derived EVs induced pro-inflammatory effects in mixed cortical cultures. Proteome analysis of these EVs revealed the presence of several pro-inflammatory proteins, including the complement protein C3. Strikingly, inhibition of EV production using GW4869 resulted in protection against acute AβO-induced cognitive decline. Further research into the underlying mechanisms of this EV secretion might open up novel therapeutic strategies to impact the pathogenesis and progression of AD. (hide) EV-METRIC 75% (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 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 Commercial method No extra separation steps Protein markers EV: CD81/ CD9/ TSG101 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Choroid plexus explants EV-harvesting Medium Serum free medium Separation Method Commercial kit qEV Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD81/ TSG101 Not detected contaminants Calnexin Detected EV-associated proteins CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample F2: 4.4E10;F3: 2.8E10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200050	1/4	Homo sapiens	Blood plasma	qEV UF	Vestad, Beate	2021	75%
Study summary Full title Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk All authors Beate Vestad, Tuula A Nyman, Malene Hove-Skovsgaard, Maria Stensland, Hedda Hoel, Anne-Marie Siebke Trøseid, Trude Aspelin, Hans Christian D Aass, Maija Puhka, Johannes R Hov, Susanne Dam Nielsen, Reidun Øvstebø, Marius Trøseid Journal Scientific Reports Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotox (show more...)HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets. (hide) EV-METRIC 75% (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 Blood plasma 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 Commercial method Ultrafiltration Protein markers EV: HSP70/ CD9 non-EV: ApoA1/ ApoB/ calnexin Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected contaminants Calnexin ELISA Detected contaminants ApoA1/ ApoB Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 146 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200050	2/4	Homo sapiens	Blood plasma	qEV UF	Vestad, Beate	2021	75%
Study summary Full title Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk All authors Beate Vestad, Tuula A Nyman, Malene Hove-Skovsgaard, Maria Stensland, Hedda Hoel, Anne-Marie Siebke Trøseid, Trude Aspelin, Hans Christian D Aass, Maija Puhka, Johannes R Hov, Susanne Dam Nielsen, Reidun Øvstebø, Marius Trøseid Journal Scientific Reports Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotox (show more...)HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets. (hide) EV-METRIC 75% (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 Blood plasma Sample origin type 2 diabetes 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 Commercial method Ultrafiltration Protein markers EV: HSP70/ CD9 non-EV: ApoA1/ ApoB/ calnexin Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected contaminants calnexin ELISA Detected contaminants ApoA1/ ApoB Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 141 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200050	3/4	Homo sapiens	Blood plasma	qEV UF	Vestad, Beate	2021	75%
Study summary Full title Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk All authors Beate Vestad, Tuula A Nyman, Malene Hove-Skovsgaard, Maria Stensland, Hedda Hoel, Anne-Marie Siebke Trøseid, Trude Aspelin, Hans Christian D Aass, Maija Puhka, Johannes R Hov, Susanne Dam Nielsen, Reidun Øvstebø, Marius Trøseid Journal Scientific Reports Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotox (show more...)HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets. (hide) EV-METRIC 75% (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 Blood plasma Sample origin HIV 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 Commercial method Ultrafiltration Protein markers EV: HSP70/ CD9 non-EV: ApoA1/ ApoB/ calnexin Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected contaminants calnexin ELISA Detected contaminants ApoA1/ ApoB Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 154 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200050	4/4	Homo sapiens	Blood plasma	qEV UF	Vestad, Beate	2021	75%
Study summary Full title Plasma extracellular vesicles in people living with HIV and type 2 diabetes are related to microbial translocation and cardiovascular risk All authors Beate Vestad, Tuula A Nyman, Malene Hove-Skovsgaard, Maria Stensland, Hedda Hoel, Anne-Marie Siebke Trøseid, Trude Aspelin, Hans Christian D Aass, Maija Puhka, Johannes R Hov, Susanne Dam Nielsen, Reidun Øvstebø, Marius Trøseid Journal Scientific Reports Abstract HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotox (show more...)HIV and type 2 diabetes (T2D) are both associated with gut microbiota alterations, low-grade endotoxemia and increased cardiovascular risk. We investigated the potential role of plasma extracellular vesicles (EVs) in relation to these processes. Plasma EVs were isolated by size exclusion chromatography in fasting individuals with HIV and T2D (n = 16), T2D only (n = 14), HIV only (n = 20) or healthy controls (n = 19), and characterized by transmission electron microscopy, western blot, nanoparticle tracking analysis and quantitative proteomics. The findings were compared to gut microbiota alterations, lipopolysaccharide levels and cardiovascular risk profile. Individuals with concomitant HIV and T2D had higher plasma EV concentration, which correlated closely with plasma lipopolysaccharides, triglycerides and Framingham score, but not with gut microbiota alterations. Proteomic analyses identified 558 human proteins, largely related to cardiometabolic disease genes and upstream regulation of inflammatory pathways, including IL-6 and IL-1β, as well as 30 bacterial proteins, mostly from lipopolysaccharide-producing Proteobacteria. Our study supports that EVs are related to microbial translocation processes in individuals with HIV and T2D. Their proteomic content suggests a contributing role in low-grade inflammation and cardiovascular risk development. The present approach for exploring gut-host crosstalk can potentially identify novel diagnostic biomarkers and therapeutic targets. (hide) EV-METRIC 75% (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 Blood plasma Sample origin HIV and type 2 diabetes 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 Commercial method Ultrafiltration Protein markers EV: HSP70/ CD9 non-EV: ApoA1/ ApoB/ calnexin Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ HSP70 Not detected contaminants calnexin ELISA Detected contaminants ApoA1/ ApoB Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 146 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200013	1/2	Bos taurus	Bovine ampulla oviductal fluid	DG qEV	Asaadi, Anise	2021	75%
Study summary Full title Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality All authors Anise Asaadi, Nima Azari Dolatabad, Hadi Atashi, Annelies Raes, Petra Van Damme, Michael Hoelker, An Hendrix, Osvaldo Bogado Pascottini, Ann Van Soom, Mojtaba Kafi, Krishna Chaitanya Pavani Journal Int J Biol Sci Abstract Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AO (show more...)Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality. (hide) EV-METRIC 75% (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 Bovine ampulla oviductal fluid 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 Density gradient Commercial method Protein markers EV: TSG101/ CD63/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim Function Sample Species Bos taurus Sample Type Bovine ampulla oviductal fluid Separation Method Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 14 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Commercial kit qEV Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 165 +/- 5.8 nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.80E+12 EM EM-type Transmission-EM Image type Close-up

	EV200013	2/2	Bos taurus	Bovine ovarian follicular fluid	DG qEV	Asaadi, Anise	2021	75%
Study summary Full title Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality All authors Anise Asaadi, Nima Azari Dolatabad, Hadi Atashi, Annelies Raes, Petra Van Damme, Michael Hoelker, An Hendrix, Osvaldo Bogado Pascottini, Ann Van Soom, Mojtaba Kafi, Krishna Chaitanya Pavani Journal Int J Biol Sci Abstract Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AO (show more...)Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality. (hide) EV-METRIC 75% (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 Bovine ovarian follicular fluid 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 Density gradient Commercial method Protein markers EV: TSG101/ CD63/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim Function Sample Species Bos taurus Sample Type Bovine ovarian follicular fluid Separation Method Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 14 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Commercial kit qEV Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 166.9 +/- 9.7 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6.44E+12 EM EM-type Transmission-EM Image type Close-up

	EV200010	1/4	Homo sapiens	HUVEC	(d)(U)C SEC UF	Kuypers, Sören	2021	75%
Study summary Full title Unsupervised Machine Learning-Based Clustering of Nanosized Fluorescent Extracellular Vesicles All authors Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani Journal Small Abstract Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease. (hide) EV-METRIC 75% (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 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 SEC UF Protein markers EV: ANXA2/ CD81/ CD9 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 90 Cell count 1.60E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins ANXA2/ CD81/ CD9 Not detected contaminants GM130 Detected EV-associated proteins CD9/ CD63/ ICAM1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100-200

	EV200010	2/4	Homo sapiens	HUVEC	(d)(U)C SEC UF	Kuypers, Sören	2021	75%
Study summary Full title Unsupervised Machine Learning-Based Clustering of Nanosized Fluorescent Extracellular Vesicles All authors Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani Journal Small Abstract Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease. (hide) EV-METRIC 75% (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 TNFalpha 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 SEC UF Protein markers EV: ANXA2/ CD81/ CD9 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 90 Cell count 1.60E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins ANXA2/ CD81/ CD9 Not detected contaminants GM130 Detected EV-associated proteins CD9/ CD63/ ICAM1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100-200

	EV200010	3/4	Homo sapiens	HUVEC	(d)(U)C SEC UF	Kuypers, Sören	2021	75%
Study summary Full title Unsupervised Machine Learning-Based Clustering of Nanosized Fluorescent Extracellular Vesicles All authors Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani Journal Small Abstract Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease. (hide) EV-METRIC 75% (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 IL1beta 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 SEC UF Protein markers EV: ANXA2/ CD81/ CD9 non-EV: GM130 Proteomics no Show all info Study aim New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 90 Cell count 1.60E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins ANXA2/ CD81/ CD9 Not detected contaminants GM130 Detected EV-associated proteins CD9/ CD63/ ICAM1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100-200

	EV200007	1/1	Homo sapiens	breast milk	DG (d)(U)C SEC	Zonneveld, Marijke	2021	75%
Study summary Full title Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses All authors Marijke I Zonneveld, Martijn J C van Herwijnen, Marcela M Fernandez-Gutierrez, Alberta Giovanazzi, Anne Marit de Groot, Marije Kleinjan, Toni M M van Capel, Alice J A M Sijts, Leonie S Taams, Johan Garssen, Esther C de Jong, Michiel Kleerebezem, Esther N M Nolte-'t Hoen, Frank A Redegeld, Marca H M Wauben Journal J Extracell Vesicles Abstract Maternal milk is nature's first functional food. It plays a crucial role in the development of the i (show more...)Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract. (hide) EV-METRIC 75% (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 breast milk 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 DG (d)(U)C SEC Protein markers EV: CD63/ Flotillin1/ CD9/ HSP70 non-EV: Lactoferrin Proteomics no EV density (g/ml) 1.06-1.19 Show all info Study aim Function Sample Species Homo sapiens Sample Type breast milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 6.5 Orientation Top-down Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900 Fraction volume (mL) 0.5 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 15 Sample volume/column (mL) 2.5 Resin type Sephadex G-100 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ HSP70 Detected contaminants Lactoferrin Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200005	1/6	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/ 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 105 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 11 Wash: time (min) 105 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Other Western Blot Detected EV-associated proteins Flotillin1/ TSG101 Not detected contaminants Calnexin Flow cytometry aspecific beads 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) 151 EV concentration Yes Particle yield particles/ml;Yes, other: 1.20E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

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