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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230973	3/4	Homo sapiens	DLD-1	(d)(U)C DC DG	Jimenez L	2023	67%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin EV-dep FBS in DMEM conditioning Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: Albumin Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.13E Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 157 EV concentration Yes Particle yield total particles in 50 microliter: 19470000000

	EV230972	1/5	Homo sapiens	DKs-8	(d)(U)C DC DG	Jimenez L	2023	67%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: Argonaute-2 Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DKs-8 EV-harvesting Medium Serum free medium Cell viability (%) 97 Cell count 224000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.130 Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Detected contaminants Argonaute-2 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment Yes RNAse type RNase cocktail RNAse concentration 5/ 200U/µL Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield as number of particles per cell per hour: 2

	EV230972	2/5	Homo sapiens	DKs-8	(d)(U)C DC DG	Jimenez L	2023	67%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Opti-MEM Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: Argonaute-2 Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DKs-8 EV-harvesting Medium Serum free medium Cell viability (%) 98 Cell count 234000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.130 Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Detected contaminants Argonaute-2 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment Yes RNAse type RNase cocktail RNAse concentration 5/ 200U/µL Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield as number of particles per cell per hour: 1.7

	EV230972	3/5	Homo sapiens	DKs-8	(d)(U)C DC DG	Jimenez L	2023	67%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: Albumin/ Argonaute-2 Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DKs-8 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 250800000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.130 Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Detected contaminants Albumin/ Argonaute-2 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment Yes RNAse type RNase cocktail RNAse concentration 5/ 200U/µL Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield as number of particles per cell per hour: 1.9

	EV230972	4/5	Homo sapiens	DLD-1	(d)(U)C DC DG	Jimenez L	2023	67%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: Argonaute-2 Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD-1 EV-harvesting Medium Serum free medium Cell viability (%) 98 Cell count 254000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.130 Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Detected contaminants Argonaute-2 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment Yes RNAse type RNase cocktail RNAse concentration 5/ 200U/µL Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield as number of particles per cell per hour: 2

	EV230972	5/5	Homo sapiens	DLD-1	(d)(U)C DC DG	Jimenez L	2023	67%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: Albumin/ Argonaute-2 Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 274000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.130 Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Detected contaminants Albumin/ Argonaute-2 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment Yes RNAse type RNase cocktail RNAse concentration 5/ 200U/µL Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield as number of particles per cell per hour: 2

	EV230969	1/1	Homo sapiens	Human Milk	(d)(U)C	Gómez-Ferrer M	2023	67%
Study summary Full title Identification of omega-3 oxylipins in human milk-derived extracellular vesicles with pro-resolutive actions in gastrointestinal inflammation. All authors Gómez-Ferrer M, Amaro-Prellezo E, Albiach-Delgado A, Ten-Domenech I, Kuligowski J, Sepúlveda P Journal Front Immunol Abstract Premature infants (PIs) are at risk of suffering necrotizing enterocolitis (NEC), and infants consum (show more...)Premature infants (PIs) are at risk of suffering necrotizing enterocolitis (NEC), and infants consuming human milk (HM) show a lower incidence than infants receiving formula. The composition of HM has been studied in depth, but the lipid content of HM-derived small extracellular vesicles (HM sEVs) remains unexplored. Identifying these molecules and their biological effects has potential for the treatment of intestinal disorders in PIs and could contribute to the development of HM-based fortified formulas. (hide) EV-METRIC 67% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Human Milk Sample origin Control condition Focus vesicles small 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: CD9/ CD63/ CD81/ HSP70/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Human Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 30 Wash: volume per pellet (ml) 24 Wash: time (min) 120 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 30 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 150-200 Used for determining EV concentration? Yes NTA Report type Size range/distribution Reported size (nm) 150-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV230965	2/2	Homo sapiens	urine	(d)(U)C ExoQuick	Tao W	2023	67%
Study summary Full title A urine extracellular vesicle lncRNA classifier for high-grade prostate cancer and increased risk of progression: A multi-center study. All authors Tao W, Wang BY, Luo L, Li Q, Meng ZA, Xia TL, Deng WM, Yang M, Zhou J, Zhang X, Gao X, Li LY, He YD Journal Cell Rep Med Abstract To construct a urine extracellular vesicle long non-coding RNA (lncRNA) classifier that can detect h (show more...)To construct a urine extracellular vesicle long non-coding RNA (lncRNA) classifier that can detect high-grade prostate cancer (PCa) of grade group 2 or greater and estimate the risk of progression during active surveillance, we identify high-grade PCa-specific lncRNAs by combined analyses of cohorts from TAHSY, TCGA, and the GEO database. We develop and validate a 3-lncRNA diagnostic model (C, being made of AC015987.1, CTD-2589M5.4, RP11-363E6.3) that can detect high-grade PCa. C shows higher accuracy than prostate cancer antigen 3 (PCA3), multiparametric magnetic resonance imaging (mpMRI), and two risk calculators (Prostate Cancer Prevention Trial [PCPT]-RC 2.0 and European Randomized Study of Screening for Prostate Cancer [ERSPC]-RC) in the training cohort (n = 350), two independent cohorts (n = 232/ n = 251), and TCGA cohort (n = 499). In the prospective active surveillance cohort (n = 182), C at diagnosis remains a powerful independent predictor for overall active surveillance progression. Thus, C is a potential biomarker for high-grade PCa and can also serve as a biomarker for improved selection of candidates for active surveillance. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type urine Sample origin prostate cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation ExoQuick Protein markers EV: CD9/ CD63/ CD81/ HSP70 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 5 Wash: time (min) 30 Wash: speed (g) 1500 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNAsequencing/ dd-PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 133 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.68E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230962	1/6	Bos bovis	bovine milk	(d)(U)C	Colella, Anna P.	2023	67%
Study summary Full title Homogenization and thermal processing reduce the concentration of extracellular vesicles in bovine milk All authors Anna P. Colella, Anuradha Prakash, John J. Miklavcic Journal Food Science and Nutrition Abstract Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Indu (show more...)Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Industrial processing treatments may affect the amount or bioactivity of EVs intrinsic to bovine milk. We investigated how the content and concentration of EVs were affected by homogenization and thermal processing of raw bovine milk. Raw milk was processed by homogenization, low-temperature (LT) heat, or pasteurization [high-temperature short-time (HTST) and ultra-high-temperature (UHT)] in a pilot processing facility. EVs were isolated from the raw and processed bovine milk using differential ultracentrifugation and quantified using a nanoparticle tracking analyzer. Bovine milk EVs were assessed for total miRNA and protein concentrations standardized to particle count using a fluorometric assay. There were 1.01 × 1010 (±3.30 × 109) EV particles per ml of bovine milk. All industrial processing treatments caused >60% decrease in EV concentration compared to the raw bovine milk. Homogenization and heat treatments independently and additively reduced the content of EVs in bovine milk. The averages of total miRNA/particle and total protein/particle concentrations were elevated threefold by low-temperature heat-processing treatment relative to HTST and UHT pasteurizations. The average diameter of EVs was reduced by 11%–16% by low temperature compared to raw milk (127 ± 13 nm). Homogenization and pasteurization indiscriminately reduce the EV concentration of bovine milk. Smaller EVs with higher protein content resist degradation when processing bovine milk at sub-pasteurization temperature. This new foundational knowledge may contribute to food product development on the preservation of EVs in processed dairy products, including bovine milk-based infant formulas that some newborns are dependent on for adequate growth and development. (hide) EV-METRIC 67% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type bovine milk Sample origin Control condition Focus vesicles 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/ CD63/ CD81/ Flotillin-1/ TSG101/ ANXA5/ EpCAM/ ICAM1 non-EV: GM130 Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Bos bovis Sample Type bovine milk 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: rotor type Type 50.2 Ti Pelleting: speed (g) 130000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin-1/ TSG101/ ANXA5/ EpCAM/ ICAM1 Not detected EV-associated proteins EpCAM Not detected contaminants GM130 Characterization: RNA analysis RNA analysis Type fluorometric for total miRNA Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 127 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.01E+10 EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV230603	1/4	Bos taurus	Blood plasma	(d)(U)C qEV Filtration	Turner N	2023	67%
Study summary Full title SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows. All authors Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD Journal J Proteome Res Abstract The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (hide) EV-METRIC 67% (93rd 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 Low-fertility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus 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: rotor type Type 50.2 Ti Pelleting: speed (g) 120,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101 Not detected EV-associated proteins GAPDH Detected contaminants Albumin Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230603	2/4	Bos taurus	Blood plasma	(d)(U)C qEV Filtration	Turner N	2023	67%
Study summary Full title SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows. All authors Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD Journal J Proteome Res Abstract The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (hide) EV-METRIC 67% (93rd 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 High-fertility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus 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: rotor type Type 50.2 Ti Pelleting: speed (g) 120,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101 Not detected EV-associated proteins GAPDH Detected contaminants Albumin Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230603	3/4	Bos taurus	Blood plasma	(d)(U)C qEV	Turner N	2023	67%
Study summary Full title SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows. All authors Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD Journal J Proteome Res Abstract The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (hide) EV-METRIC 67% (93rd 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 Low-fertility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus 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: rotor type Type 70.1 Ti Pelleting: speed (g) 100,000 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101 Not detected EV-associated proteins GAPDH Detected contaminants Albumin Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230603	4/4	Bos taurus	Blood plasma	(d)(U)C qEV	Turner N	2023	67%
Study summary Full title SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows. All authors Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD Journal J Proteome Res Abstract The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (hide) EV-METRIC 67% (93rd 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 High-fertility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus 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: rotor type Type 70.1 Ti Pelleting: speed (g) 100,000 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101 Not detected EV-associated proteins GAPDH Detected contaminants Albumin Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 112 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230602	3/3	Homo sapiens	Blood plasma	(d)(U)C	Benayas, Beatriz	2023	67%
Study summary Full title Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography All authors Beatriz Benayas, Joaquín Morales, Carolina Egea, Pilar Armisén, María Yáñez-Mó Journal J Extracell Biol Abstract Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. How (show more...)Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation. (hide) EV-METRIC 67% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD81 non-EV: ApoB/ ApoE Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma 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: rotor type AH 627 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81 Not detected contaminants ApoB/ ApoE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.00E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230374	2/13	Homo sapiens	HEK293T	(d)(U)C	Levy-Myers R	2023	67%
Study summary Full title An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function. All authors Levy-Myers R, Daudelin D, Na CH, Sockanathan S Journal Sci Adv Abstract Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the (show more...)Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the six-transmembrane protein glycerophosphodiester phosphodiesterase 3 (GDE3) regulates actin remodeling, a global EV biogenic pathway, to release an EV subtype with distinct functions. GDE3 is necessary and sufficient for releasing EVs containing annexin A1 and GDE3 from the plasma membrane via Wiskott-Aldrich syndrome protein family member 3 (WAVE3), a major regulator of actin dynamics. GDE3 is expressed in astrocytes but not neurons, yet mice lacking GDE3 [ knockout (KO)] have decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in hippocampal CA1 neurons. EVs from cultured wild-type astrocytes restore mEPSC amplitudes in KOs, while EVs from KO astrocytes or astrocytes inhibited for WAVE3 actin branching activity do not. Thus, GDE3-WAVE3 is a nonredundant astrocytic pathway that remodels actin to release a functionally distinct EV subtype, supporting the concept that independent regulation of global EV release pathways differentially regulates EV signaling within the cellular EV landscape. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ ANXA1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T 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: rotor type TLA-100.4 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Not detected EV-associated proteins ANXA1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 75 EV concentration Yes Particle yield Not reported EM EM-type Transmission-EM Image type Wide-field

	EV230374	4/13	Homo sapiens	HEK293T	(d)(U)C	Levy-Myers R	2023	67%
Study summary Full title An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function. All authors Levy-Myers R, Daudelin D, Na CH, Sockanathan S Journal Sci Adv Abstract Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the (show more...)Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the six-transmembrane protein glycerophosphodiester phosphodiesterase 3 (GDE3) regulates actin remodeling, a global EV biogenic pathway, to release an EV subtype with distinct functions. GDE3 is necessary and sufficient for releasing EVs containing annexin A1 and GDE3 from the plasma membrane via Wiskott-Aldrich syndrome protein family member 3 (WAVE3), a major regulator of actin dynamics. GDE3 is expressed in astrocytes but not neurons, yet mice lacking GDE3 [ knockout (KO)] have decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in hippocampal CA1 neurons. EVs from cultured wild-type astrocytes restore mEPSC amplitudes in KOs, while EVs from KO astrocytes or astrocytes inhibited for WAVE3 actin branching activity do not. Thus, GDE3-WAVE3 is a nonredundant astrocytic pathway that remodels actin to release a functionally distinct EV subtype, supporting the concept that independent regulation of global EV release pathways differentially regulates EV signaling within the cellular EV landscape. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin GDE3 overexpression Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ ANXA1/ GDE3 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T 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: rotor type TLA-100.4 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ ANXA1/ Actin/ GDE3 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield Not reported EM EM-type Transmission-EM Image type Wide-field

	EV230059	1/25	Homo sapiens	MCF-7	(d)(U)C	Irmer B	2023	67%
Study summary Full title Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression. All authors Irmer B, Efing J, Reitnauer LE, Angenendt A, Heinrichs S, Schubert A, Schulz M, Binder C, Tio J, Hansen U, Geyer C, Gerwing M, Bleckmann A, Menck K Journal Cell Commun Signal Abstract Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport a (show more...)Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large oncosomes 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/ Actinin-4/ CK18/ RGAP1/ CD81/ TSG101/ Syntenin-1 non-EV: GM130 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-7 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation Pelleting performed Yes Pelleting: rotor type Eppendorf A-4-81 Pelleting: speed (g) 1500 Wash: volume per pellet (ml) 1 Wash: time (min) 15 Wash: Rotor Type Sorvall Heraeus 3328 Wash: speed (g) 1500 Filtration steps Below or equal to 800/ Between 800 and 10,000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ Actinin-4/ CK18/ RGAP1 Not detected EV-associated proteins CD81/ TSG101/ Syntenin-1 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 186.2 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230059	2/25	Homo sapiens	MCF-7	(d)(U)C	Irmer B	2023	67%
Study summary Full title Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression. All authors Irmer B, Efing J, Reitnauer LE, Angenendt A, Heinrichs S, Schubert A, Schulz M, Binder C, Tio J, Hansen U, Geyer C, Gerwing M, Bleckmann A, Menck K Journal Cell Commun Signal Abstract Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport a (show more...)Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ TSG101/ Actinin-4/ CK18/ RGAP1/ CD81/ Syntenin-1 non-EV: GM130 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-7 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Filtration steps Below or equal to 800/ Between 800 and 10,000/ Equal to or above 10,000 and below 50,000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ Actinin-4/ CK18/ RGAP1 Not detected EV-associated proteins CD81/ Syntenin-1 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 158.1 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230059	3/25	Homo sapiens	MCF-7	(d)(U)C	Irmer B	2023	67%
Study summary Full title Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression. All authors Irmer B, Efing J, Reitnauer LE, Angenendt A, Heinrichs S, Schubert A, Schulz M, Binder C, Tio J, Hansen U, Geyer C, Gerwing M, Bleckmann A, Menck K Journal Cell Commun Signal Abstract Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport a (show more...)Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD81/ TSG101/ Actinin-4/ Syntenin-1/ CK18/ RGAP1 non-EV: GM130 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-7 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.3 Wash: time (min) 60 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Filtration steps Below or equal to 800/ Between 800 and 10,000/ Equal to or above 10,000 and below 50,000/ Equal to or above 100,000 and below 150,000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ TSG101/ Actinin-4/ Syntenin-1 Not detected EV-associated proteins CK18/ RGAP1 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 139 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230059	20/25	Mus musculus	4T1	(d)(U)C	Irmer B	2023	67%
Study summary Full title Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression. All authors Irmer B, Efing J, Reitnauer LE, Angenendt A, Heinrichs S, Schubert A, Schulz M, Binder C, Tio J, Hansen U, Geyer C, Gerwing M, Bleckmann A, Menck K Journal Cell Commun Signal Abstract Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport a (show more...)Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large oncosomes 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/ RGAP1/ Actinin-4/ CD81/ TSG101/ Syntenin-1 non-EV: HDAC1 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation Pelleting performed Yes Pelleting: rotor type Eppendorf A-4-81 Pelleting: speed (g) 1500 Wash: volume per pellet (ml) 1 Wash: time (min) 15 Wash: Rotor Type Sorvall Heraeus 3328 Wash: speed (g) 1500 Filtration steps Below or equal to 800/ Between 800 and 10,000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ RGAP1/ Actinin-4 Not detected EV-associated proteins CD81/ TSG101/ Syntenin-1 Not detected contaminants HDAC1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 218.2 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230059	21/25	Mus musculus	4T1	(d)(U)C	Irmer B	2023	67%
Study summary Full title Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression. All authors Irmer B, Efing J, Reitnauer LE, Angenendt A, Heinrichs S, Schubert A, Schulz M, Binder C, Tio J, Hansen U, Geyer C, Gerwing M, Bleckmann A, Menck K Journal Cell Commun Signal Abstract Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport a (show more...)Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ TSG101/ RGAP1/ Actinin-4/ CD81/ Syntenin-1 non-EV: HDAC1 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Filtration steps Below or equal to 800/ Between 800 and 10,000/ Equal to or above 10,000 and below 50,000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ RGAP1/ Actinin-4 Not detected EV-associated proteins CD81/ Syntenin-1 Not detected contaminants HDAC1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 192.8 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230059	22/25	Mus musculus	4T1	(d)(U)C	Irmer B	2023	67%
Study summary Full title Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression. All authors Irmer B, Efing J, Reitnauer LE, Angenendt A, Heinrichs S, Schubert A, Schulz M, Binder C, Tio J, Hansen U, Geyer C, Gerwing M, Bleckmann A, Menck K Journal Cell Commun Signal Abstract Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport a (show more...)Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD81/ TSG101/ Syntenin-1/ Actinin-4/ RGAP1 non-EV: HDAC1 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.3 Wash: time (min) 60 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Filtration steps Below or equal to 800/ Between 800 and 10,000/ Equal to or above 10,000 and below 50,000/ Equal to or above 100,000 and below 150,000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ TSG101/ Syntenin-1/ Actinin-4 Not detected EV-associated proteins RGAP1 Not detected contaminants HDAC1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 138.1 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230028	1/3	Homo sapiens	T lymphocyte	(d)(U)C	Li G	2023	67%
Study summary Full title miR-142-3p encapsulated in T lymphocyte-derived tissue small extracellular vesicles induces Treg function defect and thyrocyte destruction in Hashimoto's thyroiditis. All authors Li G, He L, Huang J, Liu J, Chen W, Zhong J, Wei T, Li Z, Zhu J, Lei J Journal BMC Med Abstract Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease characterized by lymphocyte inf (show more...)Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease characterized by lymphocyte infiltration that destroys thyrocyte cells. The aim of the present study was to elucidate the role and mechanisms of tissue small extracellular vesicle (sEV) microRNAs (miRNAs) in the pathogenesis of HT. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ HSP70/ TSG101/ calnexin non-EV: None Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells T lymphocyte 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: 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/ CD81/ HSP70/ TSG101 Not detected EV-associated proteins calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 130.7±48.5 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.10E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230028	2/3	Homo sapiens	T lymphocyte	(d)(U)C	Li G	2023	67%
Study summary Full title miR-142-3p encapsulated in T lymphocyte-derived tissue small extracellular vesicles induces Treg function defect and thyrocyte destruction in Hashimoto's thyroiditis. All authors Li G, He L, Huang J, Liu J, Chen W, Zhong J, Wei T, Li Z, Zhu J, Lei J Journal BMC Med Abstract Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease characterized by lymphocyte inf (show more...)Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease characterized by lymphocyte infiltration that destroys thyrocyte cells. The aim of the present study was to elucidate the role and mechanisms of tissue small extracellular vesicle (sEV) microRNAs (miRNAs) in the pathogenesis of HT. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Hashimoto thyroiditis Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ HSP70/ TSG101/ calnexin non-EV: None Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells T lymphocyte 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: 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/ CD81/ HSP70/ TSG101 Not detected EV-associated proteins calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 130.7±48.5 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.10E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230028	3/3	Homo sapiens	tissue	(d)(U)C	Li G	2023	67%
Study summary Full title miR-142-3p encapsulated in T lymphocyte-derived tissue small extracellular vesicles induces Treg function defect and thyrocyte destruction in Hashimoto's thyroiditis. All authors Li G, He L, Huang J, Liu J, Chen W, Zhong J, Wei T, Li Z, Zhu J, Lei J Journal BMC Med Abstract Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease characterized by lymphocyte inf (show more...)Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease characterized by lymphocyte infiltration that destroys thyrocyte cells. The aim of the present study was to elucidate the role and mechanisms of tissue small extracellular vesicle (sEV) microRNAs (miRNAs) in the pathogenesis of HT. (hide) EV-METRIC 67% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type tissue 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: CD63/ CD81/ HSP70/ TSG101/ calnexin non-EV: None Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: 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/ CD81/ HSP70/ TSG101 Not detected EV-associated proteins calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 127.6±61.2 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230025	1/5	Bos taurus	Milk	(d)(U)C qEV Filtration	Turner NP	2023	67%
Study summary Full title Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition. All authors Turner NP, Abeysinghe P, Sadowski P, Mitchell MD Journal Mol Nutr Food Res Abstract Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritiona (show more...)Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. (hide) EV-METRIC 67% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation qEV Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ Syn-1/ GAPDH non-EV: Albumin/ Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Bos taurus Sample Type Milk 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ TSG101/ Syntenin-1/ GAPDH Not detected contaminants Albumin/ Calnexin Proteomics database PRIDE Not detected contaminants Albumin Characterization: RNA analysis RNA analysis Type RNA-sequencing Database Vesiclepedia Proteinase treatment No RNAse treatment Yes RNAse concentration provided in kit Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.46E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230025	2/5	Homo sapiens	Milk	(d)(U)C qEV Filtration	Turner NP	2023	67%
Study summary Full title Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition. All authors Turner NP, Abeysinghe P, Sadowski P, Mitchell MD Journal Mol Nutr Food Res Abstract Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritiona (show more...)Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. (hide) EV-METRIC 67% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation qEV Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ Syn-1/ GAPDH non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Milk 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Flotillin-1/ TSG101/ Syntenin-1/ GAPDH Not detected EV-associated proteins CD81 Not detected contaminants Calnexin Proteomics database PRIDE Not detected contaminants Albumin Characterization: RNA analysis RNA analysis Type RNA-sequencing Database Vesiclepedia Proteinase treatment No RNAse treatment Yes RNAse concentration provided in kit Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.87E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230025	3/5	Bos taurus	Infant formula	(d)(U)C qEV Filtration	Turner NP	2023	67%
Study summary Full title Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition. All authors Turner NP, Abeysinghe P, Sadowski P, Mitchell MD Journal Mol Nutr Food Res Abstract Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritiona (show more...)Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. (hide) EV-METRIC 67% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Infant formula Sample origin IF 0-6 months 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 qEV Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Bos taurus Sample Type Infant formula 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101 Not detected EV-associated proteins GAPDH/ Flotillin-1/ CD81 Detected contaminants Albumin Proteomics database PRIDE Detected contaminants Albumin Characterization: RNA analysis RNA analysis Type RNA-sequencing Database Vesiclepedia Proteinase treatment No RNAse treatment Yes RNAse concentration provided in kit Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 105 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.02E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230025	4/5	Bos taurus	Infant formula	(d)(U)C qEV Filtration	Turner NP	2023	67%
Study summary Full title Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition. All authors Turner NP, Abeysinghe P, Sadowski P, Mitchell MD Journal Mol Nutr Food Res Abstract Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritiona (show more...)Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. (hide) EV-METRIC 67% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Infant formula Sample origin IF 6-12 months 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 qEV Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Bos taurus Sample Type Infant formula 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101 Not detected EV-associated proteins GAPDH/ Flotillin-1/ CD81 Detected contaminants Albumin Proteomics database PRIDE Detected contaminants Albumin Characterization: RNA analysis RNA analysis Type RNA-sequencing Database Vesiclepedia Proteinase treatment No RNAse treatment Yes RNAse concentration provided in kit Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.19E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230025	5/5	Bos taurus	Infant formula	(d)(U)C qEV Filtration	Turner NP	2023	67%
Study summary Full title Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition. All authors Turner NP, Abeysinghe P, Sadowski P, Mitchell MD Journal Mol Nutr Food Res Abstract Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritiona (show more...)Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. (hide) EV-METRIC 67% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Infant formula Sample origin IF 1 year+ 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 qEV Filtration Protein markers EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Bos taurus Sample Type Infant formula 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100,000 Filtration steps 0.2 or 0.22 ?m Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101 Not detected EV-associated proteins GAPDH/ Flotillin-1/ CD81 Detected contaminants Albumin Proteomics database PRIDE Detected contaminants Albumin Characterization: RNA analysis RNA analysis Type RNA-sequencing Database Vesiclepedia Proteinase treatment No RNAse treatment Yes RNAse concentration provided in kit Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 95 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.95E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230008	1/42	Mus musculus	EO771	(d)(U)C UF	Cocozza F	2023	67%
Study summary Full title Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells. All authors Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C Journal EMBO J Abstract Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 10k 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: CD9/ CD63/ HSP90/ MFGE8 non-EV: Argonaute-2 Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells EO771 EV-harvesting Medium Serum free medium Cell viability (%) 85 Cell count 100000000 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: rotor type MLA-80 Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 6 Wash: time (min) 16 Wash: Rotor Type MLA-80 Wash: speed (g) 10000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ HSP90/ MFGE8 Not detected contaminants Argonaute-2 Proteomics database PRIDE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 150 EV concentration Yes EM EM-type Cryo-EM Image type Wide-field

	EV230008	2/42	Mus musculus	EO771	(d)(U)C UF	Cocozza F	2023	67%
Study summary Full title Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells. All authors Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C Journal EMBO J Abstract Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 200k 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: Alix/ CD9/ CD63/ HSP90/ MFGE8 non-EV: Argonaute-2 Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells EO771 EV-harvesting Medium Serum free medium Cell viability (%) 85 Cell count 100000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type MLA-80 Pelleting: speed (g) 200000 Wash: volume per pellet (ml) 6 Wash: time (min) 50 Wash: Rotor Type MLA-80 Wash: speed (g) 200000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP90/ MFGE8 Not detected contaminants Argonaute-2 Proteomics database PRIDE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Distribution of sizes (%)/ Distribution of sizes (%) Reported size (nm) 20% (0-100), 70% (100-200), 10% (>200) EV concentration Yes EM EM-type Cryo-EM Image type Wide-field Report size (nm) 25-200

	EV230000	1/2	Bos taurus	Bovine Oviductal Epithelial Cell Organoids	(d)(U)C Filtration Size-exclusion chromatography (non-commercial)	Menjivar NG	2023	67%
Study summary Full title Bovine oviductal organoids: a multi-omics approach to capture the cellular and extracellular molecular response of the oviduct to heat stress. All authors Menjivar NG, Gad A, Thompson RE, Meyers MA, Hollinshead FK, Tesfaye D Journal BMC Genomics Abstract The mammalian oviduct is a complex, fibromuscular organ known for its role in orchestrating a series (show more...)The mammalian oviduct is a complex, fibromuscular organ known for its role in orchestrating a series of timely and dynamic changes to suitably support early embryogenesis. Climate change-induced heat stress (HS) is one of the largest single stressors compromising reproductive function in humans and farm animals via systemic changes in the redox status of the maternal environment, adversely affecting fertilization and early embryonic development. Oviductal organoids represent a unique 3-dimensional, biomimetic model to study the physiology of the oviduct and its subsequent impact on embryo development under various environmental conditions. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ FLOT1/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells Bovine Oviductal Epithelial Cell Organoids 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: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Total column volume (mL) 0.1 Sample volume/column (mL) 0.1 Other Name other separation method Size-exclusion chromatography (non-commercial) Characterization: Protein analysis Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ FLOT1/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Database NCBI's Gene Expression Omnibus (GEO) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 138.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.40E+10 EM EM-type Transmission-EM Image type Close-up

	EV230000	2/2	Bos taurus	Bovine Oviductal Epithelial Cell Organoids	(d)(U)C Filtration Size-exclusion chromatography (non-commercial)	Menjivar NG	2023	67%
Study summary Full title Bovine oviductal organoids: a multi-omics approach to capture the cellular and extracellular molecular response of the oviduct to heat stress. All authors Menjivar NG, Gad A, Thompson RE, Meyers MA, Hollinshead FK, Tesfaye D Journal BMC Genomics Abstract The mammalian oviduct is a complex, fibromuscular organ known for its role in orchestrating a series (show more...)The mammalian oviduct is a complex, fibromuscular organ known for its role in orchestrating a series of timely and dynamic changes to suitably support early embryogenesis. Climate change-induced heat stress (HS) is one of the largest single stressors compromising reproductive function in humans and farm animals via systemic changes in the redox status of the maternal environment, adversely affecting fertilization and early embryonic development. Oviductal organoids represent a unique 3-dimensional, biomimetic model to study the physiology of the oviduct and its subsequent impact on embryo development under various environmental conditions. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Heat stress (42 degrees celsius) Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ FLOT1/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells Bovine Oviductal Epithelial Cell Organoids 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: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Total column volume (mL) 0.1 Sample volume/column (mL) 0.1 Other Name other separation method Size-exclusion chromatography (non-commercial) Characterization: Protein analysis Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ FLOT1/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Database NCBI's Gene Expression Omnibus (GEO) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 138.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.00E+10 EM EM-type Transmission-EM Image type Close-up

	EV220412	1/1	Bos taurus	ovary-derived granulosa cells	(d)(U)C Filtration	Menjivar, Nico G	2023	67%
Study summary Full title Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos All authors Nico G. Menjivar, Ahmed Gad, Samuel Gebremedhn, Soham Ghosh and Dawit Tesfaye Journal Front Cell Dev Biol Abstract Climate change-induced global warming results in rises in body temperatures above normal physiologic (show more...)Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes’ thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ TSG101 non-EV: CYCS Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells ovary-derived granulosa cells EV-harvesting Medium EV-depleted medium Cell count 250000 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3.5 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101 Not detected contaminants CYCS Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 137.75 EV concentration Yes Particle yield as numer of particles per mililiter: 1.02E+11 EM EM-type Transmission-EM Image type Close-up Report size (nm) 137.75

	EV220412	2/1	Bos taurus	ovary-derived granulosa cells	(d)(U)C Filtration	Menjivar, Nico G	2023	67%
Study summary Full title Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos All authors Nico G. Menjivar, Ahmed Gad, Samuel Gebremedhn, Soham Ghosh and Dawit Tesfaye Journal Front Cell Dev Biol Abstract Climate change-induced global warming results in rises in body temperatures above normal physiologic (show more...)Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes’ thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Heat stress Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ TSG101 non-EV: CYCS Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells ovary-derived granulosa cells EV-harvesting Medium EV-depleted medium Cell count 250000 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 Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3.5 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101 Not detected contaminants CYCS Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 147.95 EV concentration Yes Particle yield as numer of particles per mililiter: 1.51E+11 EM EM-type Transmission-EM Image type Close-up Report size (nm) 147.95

	EV220409	1/2	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers ME	2023	67%
Study summary Full title Life stage-specific glycosylation of extracellular vesicles from schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL. All authors Kuipers ME, Nguyen DL, van Diepen A, Mes L, Bos E, Koning RI, Nolte-'t Hoen ENM, Smits HH, Hokke CH Journal Front Mol Biosci Abstract Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released para (show more...)Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host. (hide) EV-METRIC 67% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP2 non-EV: None Proteomics no EV density (g/ml) 1.09-1.18 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 41.60% Total gradient volume, incl. sample (mL) 2.113 Sample volume (mL) 0.293 Orientation Bottom-up Speed (g) 166,18 Duration (min) 120 Fraction volume (mL) 0.1 Fraction processing Centrifugation Pelleting: volume per fraction 2.75 Pelleting: speed (g) 187,813 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) yes, per 100 adult worms Western Blot Antibody details provided? No Detected EV-associated proteins S. mansoni TSP2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-250 EV concentration Yes Particle yield number per 100 adult worms EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 30-180 Extra information All data on protein concentration and NTA of the schistosomula EVs can be found in EV track ID EV190032. More information on the protocol for the cryo EM of this publication can be found in EV track ID EV220119. Furthermore, we performed glycomics, western blots targeting glycan structures, and lectin blots of the adult worm EVs. The western blots targeting glycan structures were also performed on the schistosomula EV (glycomics on these EV are linked to EV track ID EV190032).

	EV220366	1/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.50E+11 EM EM-type Transmission-EM Image type Wide-field

	EV220366	3/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Ionomycin 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/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.50E+11 EM EM-type Transmission-EM Image type Wide-field

	EV220366	4/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin compound 634 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/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.00E+11 EM EM-type Transmission-EM Image type Wide-field

	EV220318	1/4	Mus musculus	CAD5	(d)(U)C	Jakub Soukup	2023	67%
Study summary Full title Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles All authors Jakub Soukup, Tibor Moško, Sami Kereïche, Karel Holada Journal Biochem Pharmacol Abstract Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans (show more...)Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Prion infected cell culture Focus vesicles large extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD9/ CD63/ HSP70/ TSG101/ Integrin-beta1/ CD81/ PrP non-EV: Calnexin Proteomics no Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells CAD5 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h 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 Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 19827 Wash: volume per pellet (ml) 13.5 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 19556.1 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ TSG101/ Integrin-beta1/ PrP Not detected EV-associated proteins CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Extra information We have isolated and analysed Large and Small extracellular vesicles from the same conditioned medium aiming to separate them and use them for infection. Large EVs supposed to be above 70 nm in size and enriched in Beta 1 integrin while Small EVs are 30-150 nm and depleted in Beta 1 integrin. Secondary cell culture served as control.

	EV220318	2/4	Mus musculus	CAD5	(d)(U)C DC	Jakub Soukup	2023	67%
Study summary Full title Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles All authors Jakub Soukup, Tibor Moško, Sami Kereïche, Karel Holada Journal Biochem Pharmacol Abstract Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans (show more...)Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Prion infected cell culture Focus vesicles small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ CD63/ HSP70/ TSG101/ CD81/ PrP non-EV: Calnexin/ Integrin-beta1 Proteomics no Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells CAD5 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 112398.1 Wash: volume per pellet (ml) 13.5 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 110862.3 Density cushion Density medium Sucrose Sample volume 11 Cushion volume 2.5 Density of the cushion 40% Centrifugation time 70 Centrifugation speed 110862.3 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ TSG101/ PrP Not detected EV-associated proteins CD81 Detected contaminants Integrin-beta1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Report size (nm) 79

	EV220304	27/30	NA	NA	DG UF SEC (non-commercial)	Dhondt B	2023	67%
Study summary Full title Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. All authors Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide) EV-METRIC 67% (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 NA Sample origin NA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin Proteomics yes EV density (g/ml) 1.09-1.10 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods Sample Species NA Sample Type NA Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay Proteomics database ProteomeXchange Detected contaminants Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins Not detected contaminants Albumin/ GM130/ PMP70/ Prohibitin Characterization: RNA analysis RNA analysis Type RNA -sequencing Database BioProject Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210141	1/5	Homo sapiens	human umbilical vein endothelial cells	IAF Ultrafiltratrion (d)(U)C	Zhao F	2023	67%
Study summary Full title Extracellular vesicles from Zika virus-infected cells display viral E protein that binds ZIKV-neutralizing antibodies to prevent infection enhancement. All authors Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G Journal EMBO J Abstract Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Immunoaffinity capture (non-commercial) Ultrafiltratrion (Differential) (ultra)centrifugation Protein markers EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9 non-EV: Capsid/ E/ LC3/ Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human umbilical vein endothelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 2,00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 100 kDa Membrane type Regenerated cellulose Immunoaffinity capture Selected surface protein(s) CD9 Other Name other separation method Ultrafiltratrion 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 CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81 Detected contaminants Capsid/ E Not detected contaminants LC3/ 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 EM EM-type Transmission-EM/ Cryo-EM Image type Wide-field Report size (nm) 100

	EV210077	4/6	Homo sapiens	Serum	(d)(U)C	Lapitz A	2023	67%
Study summary Full title Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis and prognostication of cholangiocarcinoma. All authors Lapitz A, Azkargorta M, Milkiewicz P, Olaizola P, Zhuravleva E, Grimsrud MM, Schramm C, Arbelaiz A, O'Rourke CJ, La Casta A, Milkiewicz M, Pastor T, Vesterhus M, Jimenez-Agüero R, Dill MT, Lamarca A, Valle JW, Macias RIR, Izquierdo-Sanchez L, Castaño YP, Caballero-Camino FJ, Riaño I, Krawczyk M, Ibarra C, Bustamante J, Nova-Camacho LM, Falcon-Perez JM, Elortza F, Perugorria MJ, Andersen JB, Bujanda L, Karlsen TH, Folseraas T, Rodrigues PM, Banales JM Journal J Hepatol Abstract Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early d (show more...)Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early diagnostic methods, especially important for individuals at high-risk (i.e., primary sclerosing cholangitis (PSC)). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). (hide) EV-METRIC 67% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin CCA Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ CRP/ OIT3 non-EV: GRP78 Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 3 Wash: time (min) 75 Wash: Rotor Type TLA-110 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ CRP/ OIT3 Not detected contaminants GRP78 Proteomics database Yes: PRIDE. Orbitrap cohort: P Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 198 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV230981	3/6	Mus musculus	Blood plasma	(d)(U)C DG qEVoriginal/70nm	André-Grégoire G	2023	63%
Study summary Full title Isolating plasma extracellular vesicles from mouse blood using size-exclusion chromatography, density gradient, and ultracentrifugation. All authors André-Grégoire G, Roux Q, Gavard J Journal STAR Protoc Abstract Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological co (show more...)Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).. (hide) EV-METRIC 63% (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient qEVoriginal/70nm Protein markers EV: Alix/ CD9 non-EV: Albumin/ ApoB Proteomics no EV density (g/ml) 1.085-1.11 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100,000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: speed (g) 100,000 Commercial kit qEVoriginal/70nm Other Name other separation method qEVoriginal/70nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9 Not detected contaminants Albumin/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230602	1/3	Homo sapiens	SK-MEL-147	(d)(U)C UF SEC (non-commercial)	Benayas, Beatriz	2023	63%
Study summary Full title Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography All authors Beatriz Benayas, Joaquín Morales, Carolina Egea, Pilar Armisén, María Yáñez-Mó Journal J Extracell Biol Abstract Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. How (show more...)Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation. (hide) EV-METRIC 63% (93rd 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 Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ TSG101/ Syntenin non-EV: Calnexin/ VDAC/ ApoB 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 SK-MEL-147 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Cell count 25000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Resins from Agarose Bead Technologies and Cytiva Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101/ Syntenin Not detected contaminants Calnexin/ VDAC/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.00E+09 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 110

	EV230602	2/3	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial)	Benayas, Beatriz	2023	63%
Study summary Full title Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography All authors Beatriz Benayas, Joaquín Morales, Carolina Egea, Pilar Armisén, María Yáñez-Mó Journal J Extracell Biol Abstract Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. How (show more...)Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation. (hide) EV-METRIC 63% (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD81 non-EV: ApoB/ ApoE Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Resins from Agarose Bead Technologies Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81 Not detected contaminants ApoB/ ApoE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230601	1/1	Escherichia coli	BL21 DE3 Delta_msbB	(d)(U)C Filtration UF ExoLutE (SL Bigen) Dialysis	Won S	2023	63%
Study summary Full title Mass-produced gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8 T cells which enables an effective combination immunotherapy with anti-PD-1. All authors Won S, Lee C, Bae S, Lee J, Choi D, Kim MG, Song S, Lee J, Kim E, Shin H, Basukala A, Lee TR, Lee DS, Gho YS Journal J Extracell Vesicles Abstract Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive (show more...)Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive bacteria to induce potent anti-tumour responses, large-scale production of bacterial EVs remains as a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs, namely, outer membrane vesicles (OMVs). By combining metal precipitation and size-exclusion chromatography, we isolated 357 mg in total protein amount of E. coli OMVs, which was equivalent to 3.93 × 10 particles (1.10 × 10 particles/μg in total protein amounts of OMVs) from 160 L of the conditioned medium. We show that these mass-produced E. coli OMVs led to complete remission of two mouse syngeneic tumour models. Further analysis of tumour microenvironment in neoantigen-expressing tumour models revealed that E. coli OMV treatment causes increased infiltration and activation of CD8 T cells, especially those of cancer antigen-specific CD8 T cells with high expression of TCF-1 and PD-1. Furthermore, E. coli OMVs showed synergistic anti-tumour activity with anti-PD-1 antibody immunotherapy, inducing substantial tumour growth inhibition and infiltration of activated cancer antigen-specific stem-like CD8 T cells into the tumour microenvironment. These data highlight the potent anti-tumour activities of mass-produced E. coli OMVs as a novel candidate for developing next-generation cancer immunotherapeutic agents. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Ultrafiltration ExoLutE (SL Bigen) Dialysis Protein markers EV: OmpA/ CD63 non-EV: FtsZ Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches) Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells BL21 DE3 Delta_msbB EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polysulfone Commercial kit ExoLutE (SL Bigen) Other Name other separation method ExoLutE (SL Bigen) Other Name other separation method Dialysis Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins OmpA Not detected EV-associated proteins CD63 Not detected contaminants FtsZ Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 20.24 NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 2.45E+10 EM EM-type Transmission-EM Image type Wide-field

	EV230062	5/6	Mus musculus	Blood plasma	(d)(U)C Filtration	Choi YY	2023	63%
Study summary Full title The miR-126-5p and miR-212-3p in the extracellular vesicles activate monocytes in the early stage of radiation-induced vascular inflammation implicated in atherosclerosis. All authors Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM Journal J Extracell Vesicles Abstract People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide) EV-METRIC 63% (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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD9/ CD63/ CD81/ CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) Not reported Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Detected contaminants Calnexin/ GM130 Flow cytometry Type of Flow cytometry conventional flow cytometry Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 Antibody details provided? No Not detected EV-associated proteins CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA -sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 85 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type conventional flow cytometry Hardware adjustment Calibration bead size 0.22/ 0.45/ 0.88/ 1.35 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: ~35000 EM EM-type Transmission-EM Image type Close-up, Wide-field

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