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Details	EV-TRACK code	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210151	1/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	100%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin2 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hiPSC (IMR90)-4 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin2 Not detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cell per 24h;Yes, other: 2.23E7 +- 1.35E7 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	2/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	100%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin2 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CPC (IMR90)-4 EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin2/ TSG101 Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cell per 24h;Yes, other: 8.27E6 +- 3.53E6 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	3/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	100%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin2 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CMi (IMR90)-4 EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin2/ TSG101 Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cell per 24h;Yes, other: 2.95E7 +- 1.19E7 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	4/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	100%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 100% (99th 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin2 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CMm (IMR90)-4 EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin2 Not detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 nm EV concentration Yes Particle yield number of particles per million cell per 24h;Yes, other: 4.10E7 +- 9.75E6 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	5/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	89%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 89% (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 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 DG (d)(U)C Filtration Protein markers EV: TSG101 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.083 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hiPSC (DF19-9-11T.H) EV-harvesting Medium Serum free medium Cell viability (%) 90 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cells per 24h;Yes, other: 1.99E7 +- 1.99E6 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	6/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	89%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 89% (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 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 DG (d)(U)C Filtration Protein markers EV: TSG101 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.083 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CPC (DF19-9-11T.H) EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cells per 24h;Yes, other: 6.43E06 +- 5.40E05 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	7/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	89%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 89% (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 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 DG (d)(U)C Filtration Protein markers EV: TSG101 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.083 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CMi (DF19-9-11T.H) EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cells per 24h;Yes, other: 3.49E07 +- 2.85E06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	8/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	89%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 89% (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 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 DG (d)(U)C Filtration Protein markers EV: TSG101 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.083 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CMm (DF19-9-11T.H) EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cells per 24h;Yes, other: 2.13E07 +- 2.25E06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210199	1/1	Homo sapiens	Urine	(d)(U)C Filtration	Correll, Vanessa	2022	78%
Study summary Full title Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis All authors Vanessa L. Correll, Joseph J. Otto, Cristina M. Risi, Brian P. Main, Paul C. Boutros, Thomas Kislinger, Vitold E. Galkin, Julius O. Nyalwidhe, O. John Semmes, Lifang Yang Journal J Extracell Vesicles Abstract The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient (show more...)The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs. (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 Urine Sample origin prostate cancer 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/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods/Biomarker/New methodological development Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 175000 Wash: volume per pellet (ml) 12.5 Wash: time (min) 130 Wash: Rotor Type SW 40 Ti Wash: speed (g) 175000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per 1E10 particles: 1.16 Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101 Detected contaminants Tamm-Horsfall protein Not detected contaminants Calnexin Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145.9 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.20E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210187	1/5	Homo sapiens	Cell culture supernatant	(d)(U)C	Swatler, Julian	2022	78%
Study summary Full title 4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells All authors Julian Swatler, Laura Turos-Korgul, Marta Brewinska-Olchowik, Sara De Biasi, Wioleta Dudka, Bac Viet Le, Agata Kominek, Salwador Cyranowski, Paulina Pilanc, Elyas Mohammadi, Dominik Cysewski, Ewa Kozlowska, Wioleta Grabowska-Pyrzewicz, Urszula Wojda, Grzegorz W Basak, Jakub Mieczkowski, Tomasz Skorski 10 , Andrea Cossarizza 11 , Katarzyna Piwocka Journal Blood advances Abstract Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosupp (show more...)Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms. (hide) EV-METRIC 78% (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 Protein markers EV: TSG101/ Alix/ CD63/ CD81 non-EV: APOA1/ GM130 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 K562 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 3.00E+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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 60 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ Alix/ CD81 Not detected contaminants APOA1/ GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 94 EV concentration Yes Particle yield Yes, as number of particles per million cells 3.00E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210002	1/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	78%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 78% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type dissociated tissues 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ Alix/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 1,50E+09 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

	EV220003	1/2	Homo sapiens	Blood plasma	DG SEC (non-commercial)	Karimi, Nasibeh	2022	75%
Study summary Full title Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - Contributions of platelet extracellular vesicles in plasma samples All authors Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer Journal J Extracell Vesicles Abstract Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide) EV-METRIC 75% (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 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 Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin non-EV: Calnexin Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 12 ml Sample volume (mL) 6 ml Orientation Top-down Rotor type SW 41 Ti Speed (g) 178,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) 27.8 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin Not detected contaminants Calnexin Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield as total number of particles in pooled SEC fractions from 6 mL of sample: 2.90e+9 Report type Size range/distribution Report size 50-70 EV-concentration No

	EV210383	1/2	Mus musculus	Serum	ExoQuick	Mkrtchian, Soiuren	2022	75%
Study summary Full title Surgical Trauma in Mice Modifies the Content of Circulating Extracellular Vesicles All authors Souren Mkrtchian, Anette Ebberyd, Rosanne E. Veerman, María Méndez-Lago, Susanne Gabrielsson, Lars I. Eriksson, and Marta Gómez-Galán Journal Front Immunol Abstract Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling respon (show more...)Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain. (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 ExoQuick Protein markers EV: CD63/ CD81/ HSP70 non-EV: Albumin/ Argonaute?2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.75 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ HSP70 Not detected contaminants Albumin Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210383	2/2	Mus musculus	Serum	IAF	Mkrtchian, Soiuren	2022	75%
Study summary Full title Surgical Trauma in Mice Modifies the Content of Circulating Extracellular Vesicles All authors Souren Mkrtchian, Anette Ebberyd, Rosanne E. Veerman, María Méndez-Lago, Susanne Gabrielsson, Lars I. Eriksson, and Marta Gómez-Galán Journal Front Immunol Abstract Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling respon (show more...)Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain. (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 Immunoaffinity capture (non-commercial) Protein markers EV: CD63/ CD81/ HSP70 non-EV: Albumin/ Argonaute?2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Separation Method Immunoaffinity capture Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.75 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ HSP70 Detected contaminants Albumin Proteomics database Yes Characterization: RNA analysis RNA analysis Type (RT)?(q)PCR/ RNA?sequencing Database NGI Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210237	1/2	Homo sapiens	Blood plasma	qEV	Gelibter S	2022	75%
Study summary Full title The impact of storage on extracellular vesicles: A systematic study. All authors Gelibter S, Marostica G, Mandelli A, Siciliani S, Podini P, Finardi A, Furlan R Journal J Extracell Vesicles Abstract Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. Wh (show more...)Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While -80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of -80°C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment/ flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that -80°C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size-charge plots. None of the tested conditions prevented the observed effects. Freeze-thaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP -mCherry ). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non-archival, samples in majority of cases. (hide) EV-METRIC 75% (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 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 qEV Protein markers EV: Alix/ Flotillin-1/ Lamp1/ ANXA1 non-EV: GM130/ ApoE/ H3 Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) particles per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ Flotillin-1/ Lamp1/ ANXA1 Not detected contaminants GM130/ ApoE/ H3 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 50-350 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00e+11 EM EM-type Immuno-EM EM protein CD63 Image type Wide-field

	EV200029	1/2	Bos taurus	Bovine embryo culture medium	qEV	Pavani KC	2022	75%
Study summary Full title Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. All authors Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K Journal Proc Natl Acad Sci U S A Abstract SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (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 embryo culture medium 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 Protein markers EV: TSG101/ CD63/ CD9 non-EV: AGO2/ APOA1 Proteomics no Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Bovine embryo culture medium Separation Method Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101 Detected contaminants APOA1 Not detected contaminants AGO2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 94.5±1.7 E08 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200029	2/2	Bos taurus	Bovine embryo culture medium	qEV	Pavani KC	2022	75%
Study summary Full title Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. All authors Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K Journal Proc Natl Acad Sci U S A Abstract SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (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 embryo culture medium 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 Protein markers EV: TSG101/ CD63/ CD9 non-EV: AGO2/ APOA1 Proteomics no Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Bovine embryo culture medium Separation Method Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101 Detected contaminants APOA1 Not detected contaminants AGO2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 76.5±8.2 E08 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV210187	3/5	Homo sapiens	Cell culture supernatant	(d)(U)C	Swatler, Julian	2022	67%
Study summary Full title 4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells All authors Julian Swatler, Laura Turos-Korgul, Marta Brewinska-Olchowik, Sara De Biasi, Wioleta Dudka, Bac Viet Le, Agata Kominek, Salwador Cyranowski, Paulina Pilanc, Elyas Mohammadi, Dominik Cysewski, Ewa Kozlowska, Wioleta Grabowska-Pyrzewicz, Urszula Wojda, Grzegorz W Basak, Jakub Mieczkowski, Tomasz Skorski 10 , Andrea Cossarizza 11 , Katarzyna Piwocka Journal Blood advances Abstract Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosupp (show more...)Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms. (hide) EV-METRIC 67% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101 non-EV: APOA1/ GM130 Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MOLM-14 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 2.80E+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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 60 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants APOA1/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 95 EV concentration Yes Particle yield Yes, as number of particles per million cells 5.00E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	1/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin 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 Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No 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 Mean Reported size (nm) 102.9 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.17E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	2/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAFL 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 Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No 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 Mean Reported size (nm) 113.3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.34E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	3/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NASH 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 Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No 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 Mean Reported size (nm) 110.1 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.73E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210002	2/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	67%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 67% (33rd 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 dissociated tissues Sample origin BRAFV600E-induced tissue (Doxycyxline 2*24h) 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ Alix/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 3,30E+09 EM EM-type Transmission-EM Image type Close-up

	EV210002	3/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	67%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 67% (33rd 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 dissociated tissues Sample origin BRAFV600E-induced tissue (Doxycyxline4*24h) 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ Alix/ CD9/ CD63/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 1,20E+10 EM EM-type Transmission-EM Image type Close-up

	EV200174	1/2	Bos taurus	1% skim milk	(d)(U)C	Sukreet, Sonal	2022	67%
Study summary Full title Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-defined Rodent Diet All authors Sonal Sukreet, Camila Pereira Braga, Thuy T An, Jiri Adamec, Juan Cui, Janos Zempleni Journal J Nutr Abstract Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumptio (show more...)Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumption of an exosome and RNA-depleted (ERD) diet elicited phenotypes compared to controls fed an exosome and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and non-ultrasonicated milk, respectively, for casein in the AIN-93G formulation. Objective: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. Methods: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation (USE); controls were not ultrasonicated (NSE). Exosome count, size and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids and proteins were analyzed by RNA-sequencing and mass spectrometry. Intestinal transport, bioavailability and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells and C57BL/6J mice (n = 3; age 6 - 8 weeks). Results: The exosome count was 76 ± 22% lower in USE compared to NSE (P < 0.05). Ultrasonication caused a degradation of up to 100% of microRNAs. USE and NSE contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected total of 525 and 484 proteins in USE and NSE. The uptake of USEs decreased by 46 ± 30% and 40 ± 27% compared to NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). Conclusions: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells and C57BL/6J mice and causes a near-complete degradation of microRNA cargos. (hide) EV-METRIC 67% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 1% skim milk Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation No extra separation steps Protein markers EV: TSG101/ Alix/ CD63/ CD9/ CD81 non-EV: Beta-integrin/ lactalbumin/ Histone-H3 Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type 1% skim milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Fiberlite-F37L-8x100 rotor Pelleting: speed (g) 120,000 Wash: volume per pellet (ml) 30 mL Wash: time (min) 90 Wash: Rotor Type Fiberlite-F37L-8x100 rotor Wash: speed (g) 120,000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA;Qubit Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Detected contaminants lactalbumin Not detected contaminants Beta-integrin/ Histone-H3 Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 117+/-44 EV concentration Yes Particle yield Yes, per milliliter of starting sample 5.90E+11 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

	EV200174	2/2	Bos taurus	1% skim milk	(d)(U)C	Sukreet, Sonal	2022	67%
Study summary Full title Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-defined Rodent Diet All authors Sonal Sukreet, Camila Pereira Braga, Thuy T An, Jiri Adamec, Juan Cui, Janos Zempleni Journal J Nutr Abstract Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumptio (show more...)Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumption of an exosome and RNA-depleted (ERD) diet elicited phenotypes compared to controls fed an exosome and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and non-ultrasonicated milk, respectively, for casein in the AIN-93G formulation. Objective: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. Methods: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation (USE); controls were not ultrasonicated (NSE). Exosome count, size and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids and proteins were analyzed by RNA-sequencing and mass spectrometry. Intestinal transport, bioavailability and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells and C57BL/6J mice (n = 3; age 6 - 8 weeks). Results: The exosome count was 76 ± 22% lower in USE compared to NSE (P < 0.05). Ultrasonication caused a degradation of up to 100% of microRNAs. USE and NSE contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected total of 525 and 484 proteins in USE and NSE. The uptake of USEs decreased by 46 ± 30% and 40 ± 27% compared to NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). Conclusions: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells and C57BL/6J mice and causes a near-complete degradation of microRNA cargos. (hide) EV-METRIC 67% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 1% skim milk Sample origin Sonicated milk Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD81/ Alix/ TSG101/ CD63/ CD9 non-EV: Beta-integrin/ lactalbumin/ Histone H3 Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type 1% skim milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Fiberlite-F37L-8x100 rotor Pelleting: speed (g) 120,000 Wash: volume per pellet (ml) 30 mL Wash: time (min) 90 Wash: Rotor Type Fiberlite-F37L-8x100 rotor Wash: speed (g) 120,000 Characterization: Protein analysis Protein Concentration Method BCA;Qubit Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected EV-associated proteins CD81/ Alix Detected contaminants lactalbumin Not detected contaminants Beta-integrin/ Histone H3 Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 218+/-99 EV concentration Yes Particle yield Yes, per milliliter of starting sample 1.40E+11 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

	EV200112	1/2	Homo sapiens	Cell culture supernatant	(d)(U)C	Giraud, Romain	2022	67%
Study summary Full title Tracking Radiolabeled Endothelial Microvesicles Predicts Their Therapeutic Efficacy: A Proof-of-Concept Study in Peripheral Ischemia Mouse Model Using SPECT/CT Imaging All authors Romain Giraud, Anaïs Moyon, Stéphanie Simoncini, Anne-Claire Duchez, Vincent Nail, Corinne Chareyre, Ahlem Bouhlel, Laure Balasse, Samantha Fernandez, Loris Vallier, Guillaume Hache, Florence Sabatier, Françoise Dignat-George, Romaric Lacroix, Benjamin Guillet, Philippe Garrigue Journal Pharmaceutics Abstract Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as b (show more...)Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection. (hide) EV-METRIC 67% (92nd 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 No extra separation steps Protein markers EV: CD51/ CD63/ CD81/ CD31/ CD146/ caveolin/ ICAM1/ integrin beta 3 non-EV: Albumin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 70000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type JA-30.50 Wash: speed (g) 70000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD51/ caveolin/ integrin-beta3/ CD81 Not detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD146/ ICAM1/ CD31 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 434.5 EM EM-type Transmission-EM Image type Close-up Report size (nm) 434

	EV200112	2/2	Homo sapiens	Cell culture supernatant	(d)(U)C qEV	Giraud, Romain	2022	67%
Study summary Full title Tracking Radiolabeled Endothelial Microvesicles Predicts Their Therapeutic Efficacy: A Proof-of-Concept Study in Peripheral Ischemia Mouse Model Using SPECT/CT Imaging All authors Romain Giraud, Anaïs Moyon, Stéphanie Simoncini, Anne-Claire Duchez, Vincent Nail, Corinne Chareyre, Ahlem Bouhlel, Laure Balasse, Samantha Fernandez, Loris Vallier, Guillaume Hache, Florence Sabatier, Françoise Dignat-George, Romaric Lacroix, Benjamin Guillet, Philippe Garrigue Journal Pharmaceutics Abstract Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as b (show more...)Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection. (hide) EV-METRIC 67% (92nd 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 Protein markers EV: beta 3 integrin/ CD51/ CD63/ CD81/ CD31/ CD146/ caveolin/ ICAM1 non-EV: Albumin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 70000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type JA-30.50 Wash: speed (g) 70000 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD51/ caveolin/ integrin-beta3/ CD81 Not detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD31/ CD146/ ICAM1 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 434.5 EM EM-type Transmission-EM Image type Close-up Report size (nm) 407

	EV200070	1/2	Homo sapiens	Cell culture supernatant	(d)(U)C	Russell, Ashley	2022	67%
Study summary Full title Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication All authors Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication Journal Toxicol Lett. Abstract Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contribu (show more...)Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes. (hide) EV-METRIC 67% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ TSG101/ CD63/ Syntenin non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary CD14+ cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type AH-629 (36 ml) Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 245.6 nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6.89E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200070	2/2	Homo sapiens	Cell culture supernatant	(d)(U)C	Russell, Ashley	2022	67%
Study summary Full title Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication All authors Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication Journal Toxicol Lett. Abstract Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contribu (show more...)Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes. (hide) EV-METRIC 67% (92nd 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 cells exposed to cigarette smoke extract 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/ CD81/ CD63/ Syntenin non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary CD14+ cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type AH-629 (36 ml) Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Syntenin/ CD81 Not detected EV-associated proteins TSG101 Detected contaminants Calnexin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 252.45 nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 8.51E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220003	2/2	Homo sapiens	Serum	DG SEC (non-commercial)	Karimi, Nasibeh	2022	63%
Study summary Full title Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - Contributions of platelet extracellular vesicles in plasma samples All authors Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer Journal J Extracell Vesicles Abstract Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide) EV-METRIC 63% (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 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 Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin non-EV: None Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 12 ml Sample volume (mL) 6 ml Orientation Top-down Rotor type SW 41 Ti Speed (g) 178,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) 26.7 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Flotillin-1/ CD41a/ P-selectin Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield as total number of particles in pooled SEC fractions from 6 mL of sample: 2.56e+9 Report type Size range/distribution Report size 50-70 EV-concentration No

	EV210214	1/1	Homo sapiens	Cell culture supernatant	(d)(U)C UF SEC (non-commercial)	Bonifay, Amandine	2022	57%
Study summary Full title A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders All authors Amandine Bonifay, Stéphane Robert, Belinda Champagne, Paul‐Rémi Petit, Aude Eugène, Corinne Chareyre, Anne‐Claire Duchez, Mélanie Vélier, Shirley Fritz, Loris Vallier, Romaric Lacroix, Françoise Dignat‐George Journal J Extracell Vesicles Abstract Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcel (show more...)Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta. (hide) EV-METRIC 57% (88th 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 large 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ GAPDH/ Integrin-beta3-subunit/ CD15/ CD66b/ CD44/ CD11c non-EV: Albumin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary neutrophils EV-harvesting Medium Serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ GAPDH/ Integrin-beta3-subunit Detected contaminants Albumin Flow cytometry Type of Flow cytometry Cytoflex S Calibration bead size 0.1 0.16 0.22 0.24 0.3 0.5 0.9 Antibody details provided? Yes Detected EV-associated proteins CD15/ CD66b Not detected EV-associated proteins CD44/ CD11c Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Median Reported size (nm) 191 Particle analysis: flow cytometry Flow cytometer type Cytoflex S Hardware adjustment Calibration bead size 0.1 0.16 0.22 0.24 0.3 0.5 0.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220119	3/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (60th 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 whole parasite culture Sample origin adult worm 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: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.15 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% (58,7% with sample) Total gradient volume, incl. sample (mL) 11.97 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 169,044 Duration (min) >960 Fraction volume (mL) 0.997 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	4/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (60th 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 whole parasite culture Sample origin adult worm 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: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.21 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% (56.4 % with sample) Total gradient volume, incl. sample (mL) 4.25 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 169,639 Duration (min) >960 Fraction volume (mL) 0.354 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	5/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (60th 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 whole parasite culture Sample origin adult worm 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: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.21 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% (51.8 % with sample) Total gradient volume, incl. sample (mL) 1.742 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.145 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Cryo-EM Image type Wide-field

	EV220119	6/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (60th 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 whole parasite culture Sample origin adult worm 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: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.07-1.19 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 1.742 Sample volume (mL) 0.07 Orientation Top-down Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.145 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	7/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (60th 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 whole parasite culture Sample origin adult worm 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: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.25-1.11 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 15 Lowest density fraction 0.4M Highest density fraction 2.5M (2.05M with sample) Total gradient volume, incl. sample (mL) 1.86 Sample volume (mL) 0.07 Orientation Bottom-up Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.155 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220119	8/14	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers, Marije	2022	56%
Study summary Full title Optimized Protocol for the Isolation of Extracellular Vesicles from the Parasitic Worm Schistosoma mansoni with Improved Purity, Concentration, and Yield All authors Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen Journal J immunol Res Abstract In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide) EV-METRIC 56% (60th 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 whole parasite culture Sample origin adult worm 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: S. mansoni TSP-2 non-EV: None Proteomics no EV density (g/ml) 1.20-1.12 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 96,808 Density gradient Type Discontinuous Number of initial discontinuous layers 15 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 1.86 Sample volume (mL) 0.07 Orientation Top-down Rotor type TLS-55 Speed (g) 166,180 Duration (min) 120 Fraction volume (mL) 0.155 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins S. mansoni TSP-2 Characterization: Lipid analysis No Characterization: Particle analysis None

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