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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200136	4/7	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Miranda, Jezid	2018	56%
Study summary Full title Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth All authors Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon Journal Placenta Abstract Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (hide) EV-METRIC 56% (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 Blood plasma Sample origin Maternal; Fetal growth restriction Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Flotillin1/ PLAP/ CD63 non-EV: Grp94 Proteomics no EV density (g/ml) 1.12-1.188g/ml Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Bottom-up Rotor type T-8100 Speed (g) 100000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: volume per fraction 0.05 Pelleting: duration (min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins Flotillin1/ CD63/ TSG101 Not detected contaminants Grp94 ELISA Antibody details provided? No Detected EV-associated proteins PLAP Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins PLAP/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 108+/-37nm EV concentration Yes

	EV200136	6/7	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Miranda, Jezid	2018	56%
Study summary Full title Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth All authors Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon Journal Placenta Abstract Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (hide) EV-METRIC 56% (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 Blood plasma Sample origin Fetal; Fetus small for gestational age Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Flotillin1/ PLAP/ CD63 non-EV: Grp94 Proteomics no EV density (g/ml) 1.12-1.188g/ml Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Bottom-up Rotor type T-8100 Speed (g) 100000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: volume per fraction 0.05 Pelleting: duration (min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins Flotillin1/ CD63/ TSG101 Not detected contaminants Grp94 ELISA Antibody details provided? No Detected EV-associated proteins PLAP Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins PLAP/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 85 ± 17 nm EV concentration Yes

	EV200136	7/7	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Miranda, Jezid	2018	56%
Study summary Full title Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth All authors Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon Journal Placenta Abstract Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (hide) EV-METRIC 56% (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 Blood plasma Sample origin Fetal; Fetal growth restriction Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Flotillin1/ PLAP/ CD63 non-EV: Grp94 Proteomics no EV density (g/ml) 1.12-1.188g/ml Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Bottom-up Rotor type T-8100 Speed (g) 100000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: volume per fraction 0.05 Pelleting: duration (min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins Flotillin1/ CD63/ TSG101 Not detected contaminants Grp94 ELISA Antibody details provided? No Detected EV-associated proteins PLAP Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins PLAP/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 81 ± 15 nm EV concentration Yes

	EV180030	5/7	Homo sapiens	Jurkat	(d)(U)C qEV	Zhaohao Liao	2018	56%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 56% (90th 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 qEV Protein markers EV: / CD63 non-EV: AChE Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Detected contaminants AChE Other 1 Acetylcholinesterase assay Detected EV-associated proteins Detected contaminants AChE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein Other;AChE Image type Close-up, Wide-field

	EV180030	6/7	Homo sapiens	Jurkat	DG (d)(U)C	Zhaohao Liao	2018	56%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide) EV-METRIC 56% (90th 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 pseudotyped HIV-1 (NL4-3) infected 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 Protein markers EV: / CD63 non-EV: p24/ AChE Proteomics no EV density (g/ml) Not specified Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Top-down Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 60 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 35 Pelleting: duration (min) 40 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Detected contaminants p24 Not detected contaminants AChE Other 1 Acetylcholinesterase assay Detected EV-associated proteins Not detected contaminants AChE Characterization: Lipid analysis No

	EV180024	1/2	Homo sapiens	THP-1	(d)(U)C Filtration	Rice TF	2018	55%
Study summary Full title Macrophage- but not monocyte-derived extracellular vesicles induce placental pro-inflammatory responses. All authors Rice TF, Donaldson B, Bouqueau M, Kampmann B, Holder B. Journal Placenta Abstract The placenta sheds extracellular vesicles (EVs), including exosomes, into the maternal circulation. (show more...)The placenta sheds extracellular vesicles (EVs), including exosomes, into the maternal circulation. We recently demonstrated that this trafficking of EVs is bi-directional; with uptake of macrophage exosomes by the placenta inducing cytokine release. The specificity of this response is currently unknown. THP-1 cells were cultured as monocytes or differentiated to macrophages, and EVs isolated by ultra-centrifugation. The effect of EVs on human placental explants was measured by cytokine ELISA/luminex. Macrophage, but not monocyte, EVs induce the release of pro-inflammatory cytokines by the placenta. Thus, placental responses to immune cell EVs, including exosomes, reflects the phenotype of the source cell. (hide) EV-METRIC 55% (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 PMA-stimulated 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 Adj. k-factor 228.8 (pelleting) Protein markers EV: Flotillin-1/ MHC1/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS 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 F-28/50 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 228.8 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 3.24 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin-1, MHC1, TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 111 EV concentration Yes Particle yield 1400000000 Extra information Cells were checked for viability visually, but as they are adherent and impossible to get off the plate in good numbers whilst preserving viability, it is not possible to report viability accurately at time of EV collection. Both mode and median size were reported.

	EV180024	2/2	Homo sapiens	THP-1	(d)(U)C Filtration	Rice TF	2018	55%
Study summary Full title Macrophage- but not monocyte-derived extracellular vesicles induce placental pro-inflammatory responses. All authors Rice TF, Donaldson B, Bouqueau M, Kampmann B, Holder B. Journal Placenta Abstract The placenta sheds extracellular vesicles (EVs), including exosomes, into the maternal circulation. (show more...)The placenta sheds extracellular vesicles (EVs), including exosomes, into the maternal circulation. We recently demonstrated that this trafficking of EVs is bi-directional; with uptake of macrophage exosomes by the placenta inducing cytokine release. The specificity of this response is currently unknown. THP-1 cells were cultured as monocytes or differentiated to macrophages, and EVs isolated by ultra-centrifugation. The effect of EVs on human placental explants was measured by cytokine ELISA/luminex. Macrophage, but not monocyte, EVs induce the release of pro-inflammatory cytokines by the placenta. Thus, placental responses to immune cell EVs, including exosomes, reflects the phenotype of the source cell. (hide) EV-METRIC 55% (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 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 Adj. k-factor 228.8 (pelleting) Protein markers EV: Flotillin-1/ MHC1/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS 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 F-28/50 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 228.8 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 8.44 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin-1, MHC1, TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 86 EV concentration Yes Particle yield 3200000000 Extra information Cells were checked for viability visually, but as they are adherent and impossible to get off the plate in good numbers whilst preserving viability, it is not possible to report viability accurately at time of EV collection. Both mode and median size were reported.

	EV180026	1/7	Homo sapiens	SKBR3	(d)(U)C Filtration	Wenzhe Li	2018	55%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 55% (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 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 Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ HER2/ EpCAM non-EV: Calnexin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SKBR3 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63, CD81, Flotillin-1, EpCAM, HER2 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 110.2 ± 12.95 NTA Report type Size range/distribution Reported size (nm) 124.4 ± 53.3 EM EM-type Transmission-EM/ Immune-EM EM protein EpCAM,HER2 Image type Close-up, Wide-field EV concentration Yes Extra information Particle/protein ratio was determined

	EV180026	3/7	Homo sapiens	Serum	(d)(U)C Filtration	Wenzhe Li	2018	55%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 55% (91st 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 Breast 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 Adj. k-factor 104.6 (pelleting) / 104.6 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ HER2/ EpCAM non-EV: Albumin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Serum 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) 1200 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Pelleting: adjusted k-factor 104.6 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Wash: adjusted k-factor 104.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 134.8 ± 52 EM EM-type Transmission-EM Image type Close-up, Wide-field EV concentration Yes Extra information Particle/protein ratio was determined

	EV180044	1/1	Homo sapiens	PCS-500-010	(d)(U)C Filtration	Biscans A	2018	55%
Study summary Full title Hydrophobicity of Lipid-Conjugated siRNAs Predicts Productive Loading to Small Extracellular Vesicles. All authors Biscans A, Haraszti RA, Echeverria D, Miller R, Didiot MC, Nikan M, Roux L, Aronin N, Khvorova A Journal J Cell Sci Abstract Small extracellular vesicles (sEVs) show promise as natural nano-devices for delivery of therapeutic (show more...)Small extracellular vesicles (sEVs) show promise as natural nano-devices for delivery of therapeutic RNA, but efficient loading of therapeutic RNA remains a challenge. We have recently shown that the attachment of cholesterol to small interfering RNAs (siRNAs) enables efficient and productive loading into sEVs. Here, we systematically explore the ability of lipid conjugates-fatty acids, sterols, and vitamins-to load siRNAs into sEVs and support gene silencing in primary neurons. Hydrophobicity of the conjugated siRNAs defined loading efficiency and the silencing activity of siRNA-sEVs complexes. Vitamin-E-conjugated siRNA supported the best loading into sEVs and productive RNA delivery to neurons. (hide) EV-METRIC 55% (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 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 Adj. k-factor 209.7 (pelleting) / 65.69 (washing) Protein markers EV: CD81/ CD63 non-EV: Calnexin Proteomics yes Show all info Study aim Mechanism of uptake/transfer, New methodological development, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PCS-500-010 EV-harvesting Medium Serum free medium Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 209.7 Wash: time (min) 90 Wash: Rotor Type TLA-110 Wash: speed (g) 100000 Wash: adjusted k-factor 65.69 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield 3.20E+08 particles/million cells EM EM-type Transmission-EM Image type Wide-field Extra information Rotor types and NTA data added post-publication.

	EV180042	1/1	Mus musculus	RAW264.7,mouse primary osteoclasts	(d)(U)C	Ikebuchi, Yuki	2018	55%
Study summary Full title Coupling of bone resorption and formation by RANKL reverse signalling. All authors Ikebuchi Y, Aoki S, Honma M, Hayashi M, Sugamori Y, Khan M, Kariya Y, Kato G, Tabata Y, Penninger JM, Udagawa N, Aoki K, Suzuki H. Journal Nature Abstract Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of oste (show more...)Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis. (hide) EV-METRIC 55% (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 RANKL-stimulated 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 Adj. k-factor 209.7 (pelleting) / 209.7 (washing) Protein markers EV: CD81/ CD63/ CD9/ RANK non-EV: Calnexin Proteomics yes Show all info Study aim Function, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells RAW264.7,mouse primary osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 209.7 Wash: time (min) 60 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 209.7 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 2.5-3.5 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Not detected contaminants Calnexin ELISA Antibody details provided? Yes Lysis buffer provided? Yes Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up Extra information Antibody details for Western blot: rabbit anti-CD9 (Abcam, CatNo. ab92726, clone EPR2949, LotNo. GR260186-10, 1:1000), rat anti-CD63 (MBL, CatNo. D263-3, clone R5G2, LotNo. 014, 1:500), hamster anti-CD81 (Bio-Rad, CatNo. MCA1846GA, clone Eat2, LotNo. 0515, 1:500), rabbit anti-Calnexin (Abcam, CatNo. ab22595, polyclonal, LotNo. GR86850-1, 1:500)

	EV180033	3/3	Homo sapiens	MDAMB231	(d)(U)C	Busatto S	2018	55%
Study summary Full title Tangential Flow Filtration for Highly Efficient Concentration of Extracellular Vesicles from Large Volumes of Fluid. All authors Busatto S, Vilanilam G, Ticer T, Lin WL, Dickson DW, Shapiro S, Bergese P, Wolfram J1. Journal Cells Abstract Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible (show more...)Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells. (hide) EV-METRIC 55% (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 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 Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140-210 EV concentration Yes Particle yield 100000000 EM EM-type Transmission-EM Image type Wide-field

	EV180013	1/1	Homo sapiens	Blood plasma	(d)(U)C	Malin, Steven	2018	55%
Study summary Full title Low cardiorespiratory fitness is associated with higher extracellular vesicle counts in obese adults. All authors Eichner NZM, Gilbertson NM, Gaitan JM, Heiston EM, Musante L, LaSalvia S, Weltman A, Erdbrügger U, Malin SK Journal Physiol Rep Abstract Low cardiorespiratory fitness (CRF) is associated with cardiovascular disease (CVD) independent of o (show more...)Low cardiorespiratory fitness (CRF) is associated with cardiovascular disease (CVD) independent of obesity. Extracellular vesicles (EVs) are a novel target of CVD, however, it remains unknown if obese individuals with very poor fitness (VPF) have elevated EVs versus people with poor fitness (PF). Thus, we tested whether VPF was associated with greater EV subtypes in obese adults. Subjects with VPF (n = 13, VO2 peak: 15.4 ± 0.6 mL/kg/min, BMI: 34.1 ± 1.7 kg/m2 ) and PF (n = 13, VO2 peak: 25.9 ± 3.0 mL/kg/min, BMI: 32.1 ± 1.2 kg/m2 ) were compared in this cross-sectional study. After an overnight fast, AnnexinV (AV) +/- platelet (CD31+ /CD41+ ), leukocyte (CD45+ /CD41- ), and endothelial EVs (CD105+ , CD31+ /CD41- ) were analyzed from fresh platelet poor plasma via imaging flow cytometry. Body fat, blood pressure (BP), and glucose tolerance (OGTT) were also tested. Body weight, BP, and circulating glucose were similar between groups, although VPF subjects were older than PF (64.0 ± 2.1 vs. 49.8 ± 4.2 year; P < 0.05). People with VPF, compared with PF, had higher total AV- EVs (P = 0.04), AV- platelet EVs (CD31+ /CD41+ ; P = 0.006), and AV- endothelial EVs (CD31+ /CD41- ; P = 0.005) independent of age and body fat. Higher AV- platelet and endothelial EVs were associated with lower VO2 peak (r = -0.56, P = 0.006 and r = -0.55, P = 0.005, respectively). Endothelial-derived AV- /CD31+ /CD41- EVs were also related to pulse pressure (r = 0.45, P = 0.03), whereas AV- /CD105 was linked to postprandial glucose (r = 0.41, P = 0.04). VPF is associated with higher AnnexinV- total, endothelial, and platelet EVs in obese adults, suggesting that subtle differences in fitness may reduce type 2 diabetes and CVD risk through an EV-related mechanism. (hide) EV-METRIC 55% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Prediabetes 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 Adj. k-factor 113.7 (pelleting) / 379.2 (washing) Protein markers EV: TSG101/ CD105/ CD45/ CD31/ CD41/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 15 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 50000 Pelleting: adjusted k-factor 113.7 Wash: time (min) 10 Wash: Rotor Type FA-45-24-11 Wash: speed (g) 15000 Wash: adjusted k-factor 379.2 Characterization: Protein analysis PMID previous EV protein analysis Western blot Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101 Flow cytometry Type of Flow cytometry ImageStream MKII Imaging Flow Cytometer Hardware adaptation to ~100nm EV's Hardware adaptation is not needed for ImageStream. Controls included single stained EV samples, buffer only controls (collected for 2 min after filtering with a 0.1 µm filter), buffer plus antibodies Calibration bead size 0.22 Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 168 TRPS Report type Mean Reported size (nm) 182 EM EM-type Cryo-EM Image type Wide-field

	EV170014	2/4	Homo sapiens	HEK293	(d)(U)C Filtration	Dionysios C Watson	2018	55%
Study summary Full title Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes All authors Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis Journal J Extracell Vesicles Abstract The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide) EV-METRIC 55% (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 human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected 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 Adj. k-factor 111.1 (pelleting) / 111.1 (washing) Protein markers EV: hetIL-15/Lactadherin non-EV: ferritin Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium Serum free medium 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) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 111.1 Wash: time (min) 120 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 110000 Wash: adjusted k-factor 111.1 Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes Particle yield 96000000000

	EV210494	2/4	Homo sapiens	reticulocytes	(d)(U)C	Díaz-Varela M	2018	50%
Study summary Full title Proteomics study of human cord blood reticulocyte-derived exosomes. All authors Díaz-Varela M, de Menezes-Neto A, Perez-Zsolt D, Gámez-Valero A, Seguí-Barber J, Izquierdo-Useros N, Martinez-Picado J, Fernández-Becerra C, Del Portillo HA Journal Sci Rep Abstract Reticulocyte-derived exosomes (Rex), extracellular vesicles of endocytic origin, were initially disc (show more...)Reticulocyte-derived exosomes (Rex), extracellular vesicles of endocytic origin, were initially discovered as a cargo-disposal mechanism of obsolete proteins in the maturation of reticulocytes into erythrocytes. In this work, we present the first mass spectrometry-based proteomics of human Rex (HuRex). HuRex were isolated from cultures of human reticulocyte-enriched cord blood using different culture conditions and exosome isolation methods. The newly described proteome consists of 367 proteins, most of them related to exosomes as revealed by gene ontology over-representation analysis and include multiple transporters as well as proteins involved in exosome biogenesis and erythrocytic disorders. Immunoelectron microscopy validated the presence of the transferrin receptor. Moreover, functional assays demonstrated active capture of HuRex by mature dendritic cells. As only seven proteins have been previously associated with HuRex, this resource will facilitate studies on the role of human reticulocyte-derived exosomes in normal and pathological conditions affecting erythropoiesis. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: transferrin receptor/ HSP70/ stomatin/ CD71/ GAPDH non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells reticulocytes 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: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins transferrin receptor/ GAPDH/ stomatin/ HSP70 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD71 Flow cytometry specific beads Antibody details provided? Yes Antibody dilution provided? No Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 127 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD71 Image type Close-up, Wide-field Report size (nm) 70

	EV210368	1/1	Homo sapiens	FaDu	(d)(U)C Filtration UF Commercial	Abramowicz A	2018	50%
Study summary Full title Harmonization of exosome isolation from culture supernatants for optimized proteomics analysis. All authors Abramowicz A, Marczak L, Wojakowska A, Zapotoczny S, Whiteside TL, Widlak P, Pietrowska M Journal PLoS One Abstract Exosomes, the smallest subset of extracellular vesicles (EVs), have recently attracted much attentio (show more...)Exosomes, the smallest subset of extracellular vesicles (EVs), have recently attracted much attention in the scientific community. Their involvement in intercellular communication and molecular reprogramming of different cell types created a demand for a stringent characterization of the proteome which exosomes carry and deliver to recipient cells. Mass spectrometry (MS) has been extensively used for exosome protein profiling. Unfortunately, no standards have been established for exosome isolation and their preparation for MS, leading to accumulation of artefactual data. These include the presence of high-abundance exosome-contaminating serum proteins in culture media which mask low-abundance exosome-specific components, isolation methods that fail to yield "pure" vesicles or variability in protein solubilization protocols. There is an unmet need for the development of standards for exosome generation, harvesting, and isolation from cellular supernatants and for optimization of protein extraction methods before proteomics analysis by MS. In this communication, we illustrate the existing problems in this field and provide a set of recommendations that are expected to harmonize exosome processing for MS and provide the faithful picture of the proteomes carried by exosomes. The recommended workflow for effective and specific identification of proteins in exosomes released by the low number of cells involves culturing cells in medium with a reduced concentration of exosome-depleted serum, purification of exosomes by size-exclusion chromatography, a combination of different protein extraction method and removal of serum-derived proteins from the final dataset using an appropriate sample of cell-unexposed medium as a control. Application of this method allowed detection of >250 vesicle-specific proteins in exosomes from 10 mL of culture medium. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Ultrafiltration Commercial Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells FaDu EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type PES Commercial kit qEV Other Name other separation method Filtration Other Name other separation method Ultrafiltration Other Name other separation method Commercial 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/ CD81 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Distribution EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210366	1/6	Homo sapiens	Primary Lymphatic Endothelial Cells	(d)(U)C ExoQuick Filtration	Brown M	2018	50%
Study summary Full title Lymphatic exosomes promote dendritic cell migration along guidance cues. All authors Brown M, Johnson LA, Leone DA, Majek P, Vaahtomeri K, Senfter D, Bukosza N, Schachner H, Asfour G, Langer B, Hauschild R, Parapatics K, Hong YK, Bennett KL, Kain R, Detmar M, Sixt M, Jackson DG, Kerjaschki D Journal J Cell Biol Abstract Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendri (show more...)Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Exosome-rich endothelial vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Filtration Protein markers EV: CX3CL1/ CD9 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary Lymphatic Endothelial Cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CX3CL1 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 25 / 75 / 125 / 175 / 225 / 275 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 133

	EV210366	3/6	Homo sapiens	Primary Lymphatic Endothelial Cells	(d)(U)C ExoQuick Filtration	Brown M	2018	50%
Study summary Full title Lymphatic exosomes promote dendritic cell migration along guidance cues. All authors Brown M, Johnson LA, Leone DA, Majek P, Vaahtomeri K, Senfter D, Bukosza N, Schachner H, Asfour G, Langer B, Hauschild R, Parapatics K, Hong YK, Bennett KL, Kain R, Detmar M, Sixt M, Jackson DG, Kerjaschki D Journal J Cell Biol Abstract Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendri (show more...)Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin TNF- containing EV harvesting media (7ng/mL) Focus vesicles Exosome-rich endothelial vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Filtration Protein markers EV: CX3CL1/ CD9 non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary Lymphatic Endothelial Cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CX3CL1 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 25 / 75 / 125 / 175 / 225 / 275 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 83

	EV180065	1/1	Homo sapiens	HEL	DG (d)(U)C UF	Deschamps T	2018	50%
Study summary Full title Extracellular Vesicles Released by Herpes Simplex Virus 1-Infected Cells Block Virus Replication in Recipient Cells in a STING-Dependent Manner. All authors Deschamps T, Kalamvoki M Journal J Virol Abstract Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to u (show more...)Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to uninfected cells viral factors and host components, such as the stimulator of interferon genes (STING), which activates type I interferon upon foreign DNA sensing. The functions of EVs released by HSV-1-infected cells have remained unknown. Here, we describe a procedure to separate the EVs from HSV-1 virions that is based on an iodixanol/sucrose gradient. STING, along with the EV markers CD63 and CD9, was found in light-density fractions, while HSV components accumulated in heavy-density fractions. HSV-1 infection stimulated the release of EVs from the cells. The EVs derived from infected cells, but not from uninfected cells, activated innate immunity in recipient cells and suppressed viral gene expression and virus replication. Moreover, only the EVs derived from infected cells stimulated the expression of a subset of M1-type markers in recipient macrophages. Conversely, EVs derived from STING-knockdown cells failed to stimulate the expression of these M1-type markers, they activated innate immune responses to a lesser extent in recipient cells, and they did not sustain the inhibition of virus replication. These data suggest that STING from the EV donor cells contributes to the antiviral responses in cells receiving EVs from HSV-1-infected cells. Perturbations in the biogenesis of EVs by silencing CD63 or blocking the activity of the neutral spingomyelinase-2 (nSMase-2) increased the HSV-1 yields. Overall, our data suggest that the EVs released from HSV-1-infected cells negatively impact the infection and could control the dissemination of the virus.IMPORTANCE Extracellular vesicles (EVs) are released by all types of cells as they constitute major mechanism of intercellular communication and have the capacity to alter the functions of recipient cells despite their limited capacity for cargo. How the EVs released by HSV-infected cells could alter the surrounding microenvironment and influence the infection currently remains unknown. The cargo of EVs reflects the physiological state of the cells in which they were produced, so the content of EVs originating from infected cells is expected to be substantially different from that of healthy cells. Our studies indicate that the EVs released by HSV-1-infected cells carry innate immune components such as STING and other host and viral factors; they can activate innate immune responses in recipient cells and inhibit HSV-1 replication. The implication of these data is that the EVs released by HSV-1-infected cells could control HSV-1 dissemination promoting its persistence in the host. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin HSV-1 (herpes simplex 1 virus)-infected 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 UF Protein markers EV: TSG101/ CD63/ CD9/ STING/ Flotillin2 non-EV: Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer/Biogenesis/cargo sorting/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEL EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 18% Highest density fraction 6% Total gradient volume, incl. sample (mL) 12 Orientation Top-down Rotor type SW 41 Ti Speed (g) 250000 Duration (min) 120 Fraction volume (mL) 0.5 Fraction processing Dialysis and ultrafiltration Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin2/ CD9/ TSG101/ CD63/ STING Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 180 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV180033	2/3	Homo sapiens	MDAMB231	(d)(U)C Tangential flow filtration	Busatto S	2018	50%
Study summary Full title Tangential Flow Filtration for Highly Efficient Concentration of Extracellular Vesicles from Large Volumes of Fluid. All authors Busatto S, Vilanilam G, Ticer T, Lin WL, Dickson DW, Shapiro S, Bergese P, Wolfram J1. Journal Cells Abstract Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible (show more...)Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Tangential flow filtration Protein markers EV: CD81/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Other Name other separation method Tangential flow filtration Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140-210 EV concentration Yes Particle yield 10000000000 EM EM-type Transmission-EM Image type Wide-field

	EV180017	3/5	Homo sapiens	MDAMB231	DG (d)(U)C Filtration UF	Beghein E	2018	50%
Study summary Full title Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function. All authors Beghein E, Devriese D, Van Hoey E, Gettemans J Journal J Cell Sci Abstract Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Expressing cortactin nanobody (SH3 domain) 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 UF Protein markers EV: CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133 EV concentration Yes Particle yield 9.79E+09 particles/million cells

	EV180017	4/5	Homo sapiens	MDAMB231	DG (d)(U)C Filtration UF	Beghein E	2018	50%
Study summary Full title Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function. All authors Beghein E, Devriese D, Van Hoey E, Gettemans J Journal J Cell Sci Abstract Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Expressing gelsolin nanobody 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 UF Protein markers EV: CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 128 EV concentration Yes Particle yield 1.97E+09 particles/million cells

	EV180008	2/4	Homo sapiens	HCCLM3	(d)(U)C Total Exosome Isolation	Hao, Liu	2018	50%
Study summary Full title Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility All authors Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang Journal Oncogene Abstract Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Total Exosome Isolation Protein markers EV: CD81/ TSG101 non-EV: Grp94 Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCCLM3 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.76 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81, TSG101 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 133 EM EM-type Transmission-EM Image type Wide-field

	EV180008	3/4	Homo sapiens	HuH7	(d)(U)C Total Exosome Isolation	Hao, Liu	2018	50%
Study summary Full title Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility All authors Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang Journal Oncogene Abstract Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Total Exosome Isolation Protein markers EV: CD81/ TSG101 non-EV: Grp94 Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HuH7 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.2 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81, TSG101 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 148 EM EM-type Transmission-EM Image type Wide-field

	EV170014	4/4	Homo sapiens	HEK293	(d)(U)C Filtration SEC	Dionysios C Watson	2018	50%
Study summary Full title Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes All authors Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis Journal J Extracell Vesicles Abstract The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected 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 SEC Protein markers EV: hetIL-15/Lactadherin non-EV: ferritin Proteomics yes Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Size-exclusion chromatography Total column volume (mL) 24 Sample volume/column (mL) 0.5 Resin type Superdex 200 Characterization: Protein analysis Protein Concentration Method Bradford Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD63 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes Particle yield 110000000000

	EV220428	1/1	Homo sapiens	Ascites	(d)(U)C Filtration	Shenoy GN	2018	44%
Study summary Full title Exosomes Associated with Human Ovarian Tumors Harbor a Reversible Checkpoint of T-cell Responses. All authors Shenoy GN, Loyall J, Maguire O, Iyer V, Kelleher RJ, Minderman H, Wallace PK, Odunsi K, Balu-Iyer SV, Bankert RB Journal Cancer Immunol Res Abstract Nano-sized membrane-encapsulated extracellular vesicles isolated from the ascites fluids of ovarian (show more...)Nano-sized membrane-encapsulated extracellular vesicles isolated from the ascites fluids of ovarian cancer patients are identified as exosomes based on their biophysical and compositional characteristics. We report here that T cells pulsed with these tumor-associated exosomes during TCR-dependent activation inhibit various activation endpoints including translocation of NFκB and NFAT into the nucleus, upregulation of CD69 and CD107a, production of cytokines, and cell proliferation. In addition, the activation of virus-specific CD8 T cells that are stimulated with the cognate viral peptides presented in the context of class I MHC is also suppressed by the exosomes. The inhibition occurs without loss of cell viability and coincidentally with the binding and internalization of these exosomes. This exosome-mediated inhibition of T cells was transient and reversible: T cells exposed to exosomes can be reactivated once exosomes are removed. We conclude that tumor-associated exosomes are immunosuppressive and represent a therapeutic target, blockade of which would enhance the antitumor response of quiescent tumor-associated T cells and prevent the functional arrest of adoptively transferred tumor-specific T cells or chimeric antigen receptor T cells. . (hide) EV-METRIC 44% (75th 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 Ascites 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 Filtration Protein markers EV: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ EpCAM non-EV: GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 200000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Not determined Other 1 Antibody array Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ EpCAM Not detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 60-80 EM EM-type Transmission-EM Image type Wide-field

	EV220200	2/3	Homo sapiens	Urine	(d)(U)C	Chutipongtanate S	2018	44%
Study summary Full title Multiplex Biomarker Screening Assay for Urinary Extracellular Vesicles Study: A Targeted Label-Free Proteomic Approach. All authors Chutipongtanate S, Greis KD Journal Sci Rep Abstract The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detec (show more...)The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary extracellular vesicles. We developed a SWATH-MS workflow with a curated spectral library of 1,145 targets. Application of the workflow across nine replicates of three sample types (exosome-like vesicles (ELVs), microvesicles (MVs) and urine proteins (UPs)) resulted in the quantitation of 888 proteins at FDR <1%. The median-coefficient of variation of the 888 proteins in the ELV sample was 7.7%, indicating excellent reproducibility. Data analysis showed common exosome markers, (i.e. CD9, CD63, ALIX, TSG101 and HSP70) were enriched in urinary ELVs as compared to MVs and UPs. The use of a multiplex biomarker screening assay focused on ELVs was investigated, and perspectives in future applications are discussed. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles (shedding) microvesicle 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: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Centrifugation speed/ Other Used subtypes Microvesicles Characterization: Protein analysis Protein Concentration Method Pierce660 assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins HSP70 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) > 100 nm

	EV220200	3/3	Homo sapiens	Urine	(d)(U)C	Chutipongtanate S	2018	44%
Study summary Full title Multiplex Biomarker Screening Assay for Urinary Extracellular Vesicles Study: A Targeted Label-Free Proteomic Approach. All authors Chutipongtanate S, Greis KD Journal Sci Rep Abstract The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detec (show more...)The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary extracellular vesicles. We developed a SWATH-MS workflow with a curated spectral library of 1,145 targets. Application of the workflow across nine replicates of three sample types (exosome-like vesicles (ELVs), microvesicles (MVs) and urine proteins (UPs)) resulted in the quantitation of 888 proteins at FDR <1%. The median-coefficient of variation of the 888 proteins in the ELV sample was 7.7%, indicating excellent reproducibility. Data analysis showed common exosome markers, (i.e. CD9, CD63, ALIX, TSG101 and HSP70) were enriched in urinary ELVs as compared to MVs and UPs. The use of a multiplex biomarker screening assay focused on ELVs was investigated, and perspectives in future applications are discussed. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles Other/ Exosome-like Vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 EV-subtype Distinction between multiple subtypes Centrifugation speed Used subtypes Exosome-like Vesicles (Centrifugation speed Characterization: Protein analysis Protein Concentration Method Pierce660 assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ TSG101/ HSP70 Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) < 100 nm

	EV220136	1/2	Homo sapiens	MDA-MB-231	(d)(U)C Filtration:Precipitation	Huang H	2018	44%
Study summary Full title Exosomes derived from breast cancer lung metastasis subpopulations promote tumor self-seeding. All authors Huang H, Zheng X, Cai C, Yao Z, Lu S, Meng X, Miao Y, He Z, Cai C, Zou F Journal Biochem Biophys Res Commun Abstract Lung metastasis is a primary obstacle in the clinical treatment of metastatic breast cancer. Most pa (show more...)Lung metastasis is a primary obstacle in the clinical treatment of metastatic breast cancer. Most patients with lung metastasis eventually die of recurrence. Recurrence may be related to self-seeding, which occurs when circulating tumor cells re-seed into the tumors they originated from (metastasis or carcinoma in situ). Tumor-derived exosomes have been intensively revealed to promote the progression of various cancers. However, whether tumor-derived exosomes play roles in tumor self-seeding has not yet been identified. By establishing a self-seeding nude mouse model, we found that exosomes derived from MDA231-LM2 cells (subpopulations of breast cancer lung metastasis) potentiate the growth of MDA-MB-231 xenografts. More importantly, laser confocal microscopy and flow cytometry results identified that MDA231-LM2-secreted exosomes promote the seeding of MDA231-LM2 cells into MDA-MB-231 xenografts. These findings suggest MDA231-LM2-secreted exosomes as a promising target to treat breast cancer lung metastasis. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Filtration:Precipitation Protein markers EV: Alix/ CD63/ TSG101 non-EV: Catreticulin/ Lamin A/C Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 110000 Filtration steps 0.2 or 0.22 µm Other Name other separation method Filtration:Precipitation 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 Alix/ CD63/ TSG101 Not detected contaminants Catreticulin/ Lamin A/C Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 115 EM EM-type Transmission-EM Image type Close-up

	EV220136	2/2	Homo sapiens	MDA231-LM2	(d)(U)C Filtration:Precipitation	Huang H	2018	44%
Study summary Full title Exosomes derived from breast cancer lung metastasis subpopulations promote tumor self-seeding. All authors Huang H, Zheng X, Cai C, Yao Z, Lu S, Meng X, Miao Y, He Z, Cai C, Zou F Journal Biochem Biophys Res Commun Abstract Lung metastasis is a primary obstacle in the clinical treatment of metastatic breast cancer. Most pa (show more...)Lung metastasis is a primary obstacle in the clinical treatment of metastatic breast cancer. Most patients with lung metastasis eventually die of recurrence. Recurrence may be related to self-seeding, which occurs when circulating tumor cells re-seed into the tumors they originated from (metastasis or carcinoma in situ). Tumor-derived exosomes have been intensively revealed to promote the progression of various cancers. However, whether tumor-derived exosomes play roles in tumor self-seeding has not yet been identified. By establishing a self-seeding nude mouse model, we found that exosomes derived from MDA231-LM2 cells (subpopulations of breast cancer lung metastasis) potentiate the growth of MDA-MB-231 xenografts. More importantly, laser confocal microscopy and flow cytometry results identified that MDA231-LM2-secreted exosomes promote the seeding of MDA231-LM2 cells into MDA-MB-231 xenografts. These findings suggest MDA231-LM2-secreted exosomes as a promising target to treat breast cancer lung metastasis. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Filtration:Precipitation Protein markers EV: Alix/ CD63/ TSG101 non-EV: Catreticulin/ Lamin A/C Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA231-LM2 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 110000 Filtration steps 0.2 or 0.22 µm Other Name other separation method Filtration:Precipitation 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 Alix/ CD63/ TSG101 Not detected contaminants Catreticulin/ Lamin A/C Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 93 EM EM-type Transmission-EM Image type Close-up

	EV220111	1/2	Homo sapiens	Endothelial colony-forming cells	(d)(U)C Filtration	Liu W	2018	44%
Study summary Full title Distinct Anti-Fibrotic Effects of Exosomes Derived from Endothelial Colony-Forming Cells Cultured Under Normoxia and Hypoxia. All authors Liu W, Zhang H, Mai J, Chen Z, Huang T, Wang S, Chen Y, Wang J Journal Med Sci Monit Abstract BACKGROUND The therapeutic potential of endothelial colony-forming cells (ECFCs) may be impaired in (show more...)BACKGROUND The therapeutic potential of endothelial colony-forming cells (ECFCs) may be impaired in an ischemic environment. Direct injection of ECFCs is not an effective method of rescuing the ischemic heart, but exosomes derived from these cells may be a promising therapeutic tool. However, exosomes produced under normoxia and hypoxia may not be identical. Therefore, the purpose of this study was to investigate alterations in the anti-fibrotic effects of hypoxia-treated ECFC-derived exosomes and the underlying mechanism involved. MATERIAL AND METHODS ECFCs were isolated from peripheral blood and exosomes were collected from ECFCs treated with normoxia (nor-exo) or hypoxia (hyp-exo). Effects of exosomes on cardiac fibroblast activation were evaluated in vitro. MicroRNAs (miRNAs) inside the exosomes were extracted and compared using next-generation RNA sequencing. Predicted target mRNAs of miR-10b-5p were validated using a dual-luciferase reporter gene assay method. RESULTS Nor-exo significantly ameliorated cardiac fibroblast activation in vitro. These effects were attenuated in the hyp-exo-treated group. miR-10b-5p was enriched in nor-exo but not in hyp-exo. Dual-luciferase reporter gene assay found that both SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1) and histone deacetylase 4 (HDAC4) mRNAs were inhibited by miR-10b-5p. The expression of neutral sphingomyelinase 2 (N-SMase2) was decreased in hypoxia ECFCs, and this result was consistent with the changes in miR-10b-5p in hyp-exo. CONCLUSIONS Due to a reduction of miR-10b-5p, which targets the fibrotic genes Smurf1 and HDAC4, the anti-fibrotic effects of hyp-exo were abolished. (hide) EV-METRIC 44% (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 Endothelial colony-forming cells Sample origin Normoxia 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 Filtration Protein markers EV: CD9/ Alix/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Endothelial colony-forming cells Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 123000 Wash: time (min) 70 Wash: Rotor Type SW 41 Ti Wash: speed (g) 123000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Detected EV-associated proteins CD9/ Alix/ CD63 Not detected contaminants Calnexin 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 Modus Reported size (nm) 110 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report type Not Reported EV-concentration No

	EV220111	2/2	Homo sapiens	Endothelial colony-forming cells	(d)(U)C Filtration	Liu W	2018	44%
Study summary Full title Distinct Anti-Fibrotic Effects of Exosomes Derived from Endothelial Colony-Forming Cells Cultured Under Normoxia and Hypoxia. All authors Liu W, Zhang H, Mai J, Chen Z, Huang T, Wang S, Chen Y, Wang J Journal Med Sci Monit Abstract BACKGROUND The therapeutic potential of endothelial colony-forming cells (ECFCs) may be impaired in (show more...)BACKGROUND The therapeutic potential of endothelial colony-forming cells (ECFCs) may be impaired in an ischemic environment. Direct injection of ECFCs is not an effective method of rescuing the ischemic heart, but exosomes derived from these cells may be a promising therapeutic tool. However, exosomes produced under normoxia and hypoxia may not be identical. Therefore, the purpose of this study was to investigate alterations in the anti-fibrotic effects of hypoxia-treated ECFC-derived exosomes and the underlying mechanism involved. MATERIAL AND METHODS ECFCs were isolated from peripheral blood and exosomes were collected from ECFCs treated with normoxia (nor-exo) or hypoxia (hyp-exo). Effects of exosomes on cardiac fibroblast activation were evaluated in vitro. MicroRNAs (miRNAs) inside the exosomes were extracted and compared using next-generation RNA sequencing. Predicted target mRNAs of miR-10b-5p were validated using a dual-luciferase reporter gene assay method. RESULTS Nor-exo significantly ameliorated cardiac fibroblast activation in vitro. These effects were attenuated in the hyp-exo-treated group. miR-10b-5p was enriched in nor-exo but not in hyp-exo. Dual-luciferase reporter gene assay found that both SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1) and histone deacetylase 4 (HDAC4) mRNAs were inhibited by miR-10b-5p. The expression of neutral sphingomyelinase 2 (N-SMase2) was decreased in hypoxia ECFCs, and this result was consistent with the changes in miR-10b-5p in hyp-exo. CONCLUSIONS Due to a reduction of miR-10b-5p, which targets the fibrotic genes Smurf1 and HDAC4, the anti-fibrotic effects of hyp-exo were abolished. (hide) EV-METRIC 44% (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 Endothelial colony-forming cells Sample origin Hypoxia 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 Filtration Protein markers EV: CD9/ Alix/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Endothelial colony-forming cells Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 123000 Wash: time (min) 70 Wash: Rotor Type SW 41 Ti Wash: speed (g) 123000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Detected EV-associated proteins CD9/ Alix/ CD63 Not detected contaminants Calnexin 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 Modus Reported size (nm) 110 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report type Not Reported EV-concentration No

	EV220058	1/5	Homo sapiens	HCT-116	(d)(U)C	Lee CH	2018	44%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Protein markers EV: Alix/ HSP70/ TSPAN1/ LGALS3BP/ SLC1A/ CLDN7/ GPRC5A/ CD63 non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT-116 EV-harvesting Medium EV-depleted medium 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: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 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/ TSPAN1/ LGALS3BP/ SLC1A/ CLDN7/ GPRC5A/ HSP70/ Alix Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-500 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV220058	2/5	Homo sapiens	HT-29	(d)(U)C	Lee CH	2018	44%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Protein markers EV: Alix/ HSP70/ TSPAN1/ LGALS3BP/ SLC1A/ CLDN7/ GPRC5A/ CD63 non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT-29 EV-harvesting Medium EV-depleted medium 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: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 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/ TSPAN1/ LGALS3BP/ SLC1A/ CLDN7/ GPRC5A/ HSP70/ Alix Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-500 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV220046	3/5	Homo sapiens	DKO-1	DG Filtration UF dUC	Zhang Q	2018	44%
Study summary Full title Mutant KRAS Exosomes Alter the Metabolic State of Recipient Colonic Epithelial Cells. All authors Zhang Q, Jeppesen DK, Higginbotham JN, Demory Beckler M, Poulin EJ, Walsh AJ, Skala MC, McKinley ET, Manning HC, Hight MR, Schulte ML, Watt KR, Ayers GD, Wolf MM, Andrejeva G, Rathmell JC, Franklin JL, Coffey RJ Journal Cell Mol Gastroenterol Hepatol Abstract NA (show more...)NA (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG Filtration UF dUC Protein markers EV: Alix/ CD81/ GLUT-1/ Synthenin non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DKO-1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 150000 Wash: time (min) 180 Wash: speed (g) 150000 Density gradient Type Discontinuous Lowest density fraction 5% Highest density fraction 45% Orientation Bottom-up Rotor type TH-641 Speed (g) 120000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: duration (min) 240 Pelleting: rotor type SureSpin 630 Pelleting: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GLUT-1/ Synthenin/ CD81 Characterization: Lipid analysis No

	EV220042	1/4	Mus musculus	Red blood cells	(d)(U)C	Jana S	2018	44%
Study summary Full title Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles. All authors Jana S, Strader MB, Meng F, Hicks W, Kassa T, Tarandovskiy I, De Paoli S, Simak J, Heaven MR, Belcher JD, Vercellotti GM, Alayash AI Journal JCI insight Abstract The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) format (show more...)The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent β-globin posttranslational modifications, including the irreversible oxidation of βCys93 and the ubiquitination of βLys96 and βLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, βCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Townes-SS Focus vesicles microparticle 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: HO-1/ ubiquitin/ band 3 Hb/ alpha subunit of Hb/ beta subunit of Hb non-EV: None Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Red blood 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 Pelleting performed Yes Pelleting: speed (g) 25000 Wash: time (min) 60 Wash: speed (g) 25000 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 HO-1/ ubiquitin/ band 3 Hb/ alpha subunit of Hb/ beta subunit of Hb Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220042	2/4	Mus musculus	Red blood cells	(d)(U)C	Jana S	2018	44%
Study summary Full title Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles. All authors Jana S, Strader MB, Meng F, Hicks W, Kassa T, Tarandovskiy I, De Paoli S, Simak J, Heaven MR, Belcher JD, Vercellotti GM, Alayash AI Journal JCI insight Abstract The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) format (show more...)The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent β-globin posttranslational modifications, including the irreversible oxidation of βCys93 and the ubiquitination of βLys96 and βLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, βCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Townes-AA Focus vesicles microparticle 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: HO-1/ ubiquitin/ band 3 Hb/ alpha subunit of Hb/ beta subunit of Hb non-EV: None Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Red blood 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 Pelleting performed Yes Pelleting: speed (g) 25000 Wash: time (min) 60 Wash: speed (g) 25000 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 HO-1/ ubiquitin/ band 3 Hb/ alpha subunit of Hb/ beta subunit of Hb Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV220036	2/3	Canis familiaris	CYPp	(d)(U)C	Sammarco A	2018	44%
Study summary Full title Preliminary investigation of extracellular vesicles in mammary cancer of dogs and cats: Identification and characterization. All authors Sammarco A, Finesso G, Cavicchioli L, Ferro S, Caicci F, Zanetti R, Sacchetto R, Zappulli V Journal Vet Comp Oncol Abstract Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role (show more...)Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role in cell-to-cell communication. They exert pleiotropic biological functions via the horizontal transfer of bioactive molecules (DNA, RNAs, proteins, and lipids) within the tumour microenvironment and throughout the body. In human cancer, EVs are known to interfere with pathways that lead to tumour progression and are used as novel cancer biomarkers. In veterinary medicine, very little is known on cancer-derived EVs. In this study, we preliminarily characterized EVs in mammary gland cancer of dogs and cats. EVs were isolated by ultracentrifugation from canine (CYPp), feline (FMCp) and human (MCF7) mammary tumour cell lines. EVs were visualized by transmission electron microscopy (TEM), counted using nanoparticle tracking analysis (NTA) and characterized by immunogold (CD63 and Alix) and western blot (Alix and TSG101). Additionally, EV production by "donor" cells (palmtdTomato ) and uptake by "recipient" cells (GFP ) were assessed. EVs were successfully isolated from all 3 cell lines by ultracentrifugation. Membrane-bound structures (50-400 nm) were identified by TEM and were positive for both CD63 and Alix at immunogold. Western blot showed positivity of EVs to Alix and TSG101. NTA analysis detected EVs from cell culture media ranging from 1.67 to 2.56 × 10 as number of EVs/cell and from 80 to 600 nm in size. Confocal microscopy identified the presence of palmtdTomato EVs into the cytoplasm of GFP cells. This preliminary study identified and characterized canine and feline mammary tumour cell-derived EVs, opening in veterinary medicine a new interesting unexplored field with several applications and limitless potential. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Protein markers EV: TSG101/ Alix non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Canis familiaris Sample Type Cell culture supernatant EV-producing cells CYPp EV-harvesting Medium EV-depleted medium Preparation of EDS 16h at 100,000g + 0.22 um filtration/ Other preparation 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: speed (g) 100000 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 TSG101/ Alix Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per million cells: 2.56e+8 EM EM-type Immuno-EM/ Transmission-EM EM protein Other/ CD63/ Alix Image type Close-up, Wide-field

	EV210404	1/1	Homo sapiens	Corneal mesenchymal stem cells	(d)(U)C UF	Samaeekia R	2018	44%
Study summary Full title Effect of Human Corneal Mesenchymal Stromal Cell-derived Exosomes on Corneal Epithelial Wound Healing. All authors Samaeekia R, Rabiee B, Putra I, Shen X, Park YJ, Hematti P, Eslani M, Djalilian AR Journal Invest Ophthalmol Vis Sci Abstract Mesenchymal stromal cells (MSCs) have been used therapeutically to modulate inflammation and promote (show more...)Mesenchymal stromal cells (MSCs) have been used therapeutically to modulate inflammation and promote repair. Extracellular vesicles, including exosomes, have been identified as one of the important mediators. This study investigated the effect of human corneal MSC-derived exosomes on corneal epithelial wound healing. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Ultrafiltration Protein markers EV: CD81/ HSP70/ CD63/ CD9 non-EV: Calnexin/ Cytochrome c/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Corneal mesenchymal stem cells EV-harvesting Medium Serum free medium 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: speed (g) 100000 Wash: time (min) 120 Wash: speed (g) 100000 Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ HSP70/ CD81 Detected contaminants Calnexin/ GM130 Not detected contaminants Cytochrome c Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 40-280 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210378	9/10	Homo sapiens	MCF-7	(d)(U)C Filtration	Jin D	2018	44%
Study summary Full title ExoAPP: Exosome-Oriented, Aptamer Nanoprobe-Enabled Surface Proteins Profiling and Detection. All authors Jin D, Yang F, Zhang Y, Liu L, Zhou Y, Wang F, Zhang GJ Journal Anal Chem Abstract Tumor exosomes that inherit molecular markers from their parent cells are emerging as cellular "surr (show more...)Tumor exosomes that inherit molecular markers from their parent cells are emerging as cellular "surrogates" in cancer diagnostics. Molecular profiling and detection of exosomes offer a noninvasive access to the state of cancer progression, yet are still technically challenging. Here we report an exosome-oriented, aptamer nanoprobe-based profiling (ExoAPP) assay to phenotype surface proteins and quantify cancerous exosomes in a facile mix-and-detect format. Our ExoAPP interfaces graphene oxide (GO) with target-responsive aptamers to profile exosomal markers across five cell types by complementing with enzyme-assisted exosome recycling, revealing a heterogeneous pattern.This assay achieves a detection limit down to 1.6 × 10 particles/mL, lowered by several orders of magnitude over other homogeneous protocols. Such a sensitive ExoAPP assay allows for monitoring epithelial-mesenchymal transition through heterogeneous exosomes without involving cellular internalization that often occurs in GO-based cargo delivery. Using ExoAPP to analyze blood samples from prostate cancer patients, we find that target exosome can be identified by surface PSMA, suggesting their potential in clinical diagnosis. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 Filtration Protein markers EV: CD63/ EpCAM/ CEA/ PTK7/ AFP/ PSMA/ PDGF non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-7 EV-harvesting Medium Serum free medium 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: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63 Detected EV-associated proteins CD63/ EpCAM/ CEA/ PTK7/ AFP/ PSMA/ PDGF Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 126 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 90

	EV210349	2/13	Homo sapiens	NA	(d)(U)C	Coulter ME	2018	44%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 44% (80th 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 NA 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 Protein markers EV: TSG101/ CD63/ CD81/ Syntenin/ CD9/ CHMP1A/ SHH non-EV: gp96/ actin Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 5 Wash: time (min) 40 Wash: Rotor Type SW 55 Ti Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins SHH Not detected EV-associated proteins CD81/ Syntenin/ TSG101/ CD63/ CD9/ CHMP1A Not detected contaminants actin/ gp96 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210349	3/13	Homo sapiens	NA	(d)(U)C	Coulter ME	2018	44%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 44% (80th 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 NA 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 Protein markers EV: TSG101/ CD63/ CD81/ Syntenin/ CD9/ CHMP1A/ RAB18/ AXL/ TMED10 non-EV: gp96/ actin Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins SHH/ CD81/ Syntenin/ TSG101/ CD63/ CD9 Not detected EV-associated proteins CHMP1A/ RAB18/ AXL/ TMED10 Not detected contaminants actin/ gp96 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210349	9/13	Homo sapiens	NA	(d)(U)C	Coulter ME	2018	44%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 44% (80th 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 NA Sample origin CHMP1A null Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD81/ Syntenin/ TSG101/ CD63/ CD9/ CHMP1A/ SHH non-EV: gp96/ actin Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 5 Wash: time (min) 40 Wash: Rotor Type SW 55 Ti Wash: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins SHH Not detected EV-associated proteins CD81/ Syntenin/ TSG101/ CD63/ CD9/ CHMP1A Not detected contaminants actin/ gp96 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210349	10/13	Homo sapiens	NA	(d)(U)C	Coulter ME	2018	44%
Study summary Full title The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. All authors Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T, Haussler D, Jacobs FMJ, Gaudin R, Walsh CA Journal Cell Rep Abstract Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and re (show more...)Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain. (hide) EV-METRIC 44% (80th 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 NA Sample origin CHMP1A null Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD81/ Syntenin/ TSG101/ CD63/ CD9/ CHMP1A/ SHH non-EV: gp96/ actin Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type NA EV-producing cells SVG-A EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 5 Wash: time (min) 90 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins SHH Not detected EV-associated proteins CD81/ Syntenin/ TSG101/ CD63/ CD9/ CHMP1A Not detected contaminants actin/ gp96 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210072	1/5	Homo sapiens	HOS	(d)(U)C	Gong, Liangzhi	2018	44%
Study summary Full title Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1 All authors Liangzhi Gong, Qiyuan Bao, Chuanzhen Hu, Jun Wang, Qi Zhou, Li Wei, Lei Tong, Weibin Zhang, Yuhui Shen Journal Biochem Biophys Res Commun Abstract Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer (show more...)Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD81/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HOS EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Alix/ CD81 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;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) 1-250 EM EM-type Transmission-EM Image type Wide-field

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