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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210502	2/8	Homo sapiens	Primary GBM Stem Cells RN1	(d)(U)C Filtration DG	Hallal S	2019	57%
Study summary Full title Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways. All authors Hallal S, Mallawaaratchy DM, Wei H, Ebrahimkhani S, Stringer BW, Day BW, Boyd AW, Guillemin GJ, Buckland ME, Kaufman KL Journal Mol Neurobiol Abstract The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tu (show more...)The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES/CD133) and their differentiated (diff) progeny cells (NES/CD133). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary GBM Stem Cells RN1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: time (min) 180 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 240 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 142

	EV210502	3/8	Homo sapiens	Primary GBM Stem Cells RN1	(d)(U)C Filtration DG	Hallal S	2019	57%
Study summary Full title Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways. All authors Hallal S, Mallawaaratchy DM, Wei H, Ebrahimkhani S, Stringer BW, Day BW, Boyd AW, Guillemin GJ, Buckland ME, Kaufman KL Journal Mol Neurobiol Abstract The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tu (show more...)The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES/CD133) and their differentiated (diff) progeny cells (NES/CD133). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Differentiated Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary GBM Stem Cells RN1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: time (min) 180 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 240 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 117

	EV210502	4/8	Homo sapiens	Primary GBM Stem Cells WK1	(d)(U)C Filtration DG	Hallal S	2019	57%
Study summary Full title Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways. All authors Hallal S, Mallawaaratchy DM, Wei H, Ebrahimkhani S, Stringer BW, Day BW, Boyd AW, Guillemin GJ, Buckland ME, Kaufman KL Journal Mol Neurobiol Abstract The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tu (show more...)The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES/CD133) and their differentiated (diff) progeny cells (NES/CD133). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary GBM Stem Cells WK1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: time (min) 180 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 240 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 135

	EV210502	5/8	Homo sapiens	Primary GBM Stem Cells WK1	(d)(U)C Filtration DG	Hallal S	2019	57%
Study summary Full title Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways. All authors Hallal S, Mallawaaratchy DM, Wei H, Ebrahimkhani S, Stringer BW, Day BW, Boyd AW, Guillemin GJ, Buckland ME, Kaufman KL Journal Mol Neurobiol Abstract The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tu (show more...)The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES/CD133) and their differentiated (diff) progeny cells (NES/CD133). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Differentiated Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary GBM Stem Cells WK1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: time (min) 180 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 240 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 112.5

	EV210326	1/1	Spodoptera frugiperda	Midgut epithelium	(d)(U)C DG	Fuzita FJ	2019	57%
Study summary Full title Detergent-resistant domains in Spodoptera frugiperda midgut microvillar membranes and their relation to microapocrine secretion. All authors Fuzita FJ, Pimenta DC, Palmisano G, Terra WR, Ferreira C Journal Comp Biochem Physiol B Biochem Mol Biol Abstract The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named mi (show more...)The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery. (hide) EV-METRIC 57% (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 Midgut epithelium Sample origin Control condition Focus vesicles microapocrine vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.13-1.24 Show all info Study aim Function/ Identification of content (omics approaches) Sample Species Spodoptera frugiperda Sample Type Midgut epithelium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type P40ST Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 25 Highest density fraction 60 Total gradient volume, incl. sample (mL) Not reported Sample volume (mL) 2 Orientation Top-down Rotor type P40ST Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing None Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV190020	1/3	Homo sapiens	SW948	DG (d)(U)C Filtration	Kyuno, Daisuke	2019	57%
Study summary Full title Claudin7-dependent exosome-promoted reprogramming of non-metastasizing tumor cells. All authors Kyuno D, Zhao K, Schnölzer M, Provaznik J, Hackert T, Zöller M. Journal Int J Cancer Abstract Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdow (show more...)Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor-exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEX were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX. This article is protected by copyright. All rights reserved. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Protein markers EV: non-EV: Proteomics yes EV density (g/ml) 1.15-1.56 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW948 EV-harvesting Medium Serum free medium Cell viability (%) 100 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1.28 Fraction processing Centrifugation Pelleting: volume per fraction 50 Pelleting: duration (min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 50 Pelleting-wash: duration (min) 150 Pelleting-wash: speed (g) Type 45 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: RNA analysis RNA analysis Type Database Proteinase treatment RNAse treatment Characterization: Lipid analysis No

	EV190020	2/3	Rattus norvegicus	ASML	DG (d)(U)C Filtration	Kyuno, Daisuke	2019	57%
Study summary Full title Claudin7-dependent exosome-promoted reprogramming of non-metastasizing tumor cells. All authors Kyuno D, Zhao K, Schnölzer M, Provaznik J, Hackert T, Zöller M. Journal Int J Cancer Abstract Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdow (show more...)Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor-exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEX were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX. This article is protected by copyright. All rights reserved. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Protein markers EV: non-EV: Proteomics yes EV density (g/ml) 1.15-1.56 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells ASML EV-harvesting Medium Serum free medium Cell viability (%) 100 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1.28 Fraction processing Centrifugation Pelleting: volume per fraction 50 Pelleting: duration (min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 50 Pelleting-wash: duration (min) 150 Pelleting-wash: speed (g) Type 45 Ti Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

	EV180072	1/4	Homo sapiens	HCT116-TGFBR2	(d)(U)C Total Exosome Isolation UF	Fricke F	2019	57%
Study summary Full title TGFBR2‑dependent alterations of microRNA profiles in extracellular vesicles and parental colorectal cancer cells. All authors Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J. Journal Int J Oncol Abstract In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Total Exosome Isolation UF Protein markers EV: non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116-TGFBR2 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 Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type ClickSeal Biocontainment Rotor with Lid; 24 x 1.5/2.0 mL Tubes Pelleting: speed (g) 21100 Ultra filtration Cut-off size (kDa) 10000 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: RNA analysis RNA analysis Type RNA sequencing;(RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 128 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV180072	2/4	Homo sapiens	HCT116-TGFBR2	(d)(U)C Total Exosome Isolation UF	Fricke F	2019	57%
Study summary Full title TGFBR2‑dependent alterations of microRNA profiles in extracellular vesicles and parental colorectal cancer cells. All authors Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J. Journal Int J Oncol Abstract In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin genetically modified cell line Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Total Exosome Isolation UF Protein markers EV: non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116-TGFBR2 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 Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type ClickSeal Biocontainment Rotor with Lid Thermo Scientific Pelleting: speed (g) 21100 Ultra filtration Cut-off size (kDa) 10000 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing;(RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 125 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV180060	1/2	Homo sapiens	NA	(d)(U)C SEC UF	Benedikter BJ	2019	57%
Study summary Full title Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles. All authors Benedikter BJ, Bouwman FG, Heinzmann ACA, Vajen T, Mariman EC, Wouters EFM, Savelkoul PHM, Koenen RR, Rohde GGU, van Oerle R, Spronk HM, Stassen FRM Journal J Extracell Vesicles Abstract Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed (show more...)Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. (hide) EV-METRIC 57% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type NA Sample origin Control condition Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C SEC UF Protein markers EV: CD63/ MFGE8/ CD81/ TF/ HSP70/ CD9 non-EV: Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type NA EV-producing cells BEAS-2B EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ CD81/ MFGE8/ CD63 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins TF/ CD63/ CD81/ CD9 Proteomics database Yes Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 80-250 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 70

	EV180060	2/2	Homo sapiens	NA	(d)(U)C SEC UF	Benedikter BJ	2019	57%
Study summary Full title Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles. All authors Benedikter BJ, Bouwman FG, Heinzmann ACA, Vajen T, Mariman EC, Wouters EFM, Savelkoul PHM, Koenen RR, Rohde GGU, van Oerle R, Spronk HM, Stassen FRM Journal J Extracell Vesicles Abstract Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed (show more...)Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. (hide) EV-METRIC 57% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type NA Sample origin 1% cigarette smoke extract Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C SEC UF Protein markers EV: TF/ CD81/ CD63/ CD9 non-EV: Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type NA EV-producing cells BEAS-2B EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Bradford Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins TF/ CD63/ CD81/ CD9 Proteomics database Yes Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 80-250 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 65

	EV180009	3/3	Danio rerio	Dissociated embryo	(d)(U)C IAF	Frederik J.Verweij	2019	57%
Study summary Full title Live Tracking of Inter-organ Communication by Endogenous Exosomes In Vivo All authors Frederik J.Verweij, Celine Revenu, Guillaume Arras, Florent Dingli, Damarys Loew, Michiel D.Pegtel, Gautier Follain, Guillaume Allio, Jacky G.Goetz, Pascale Zimmermann, Philippe Herbomel, Filippo Del Bene, GraçaRaposo, Guillaumevan Niel Journal Cell Press Abstract Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physio (show more...)Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physiological relevance remains challenging due to a lack of appropriate model organisms. Here, we developed an in vivo model to study EV function by expressing CD63-pHluorin in zebrafish embryos. A combination of imaging methods and proteomic analysis allowed us to study biogenesis, composition, transfer, uptake, and fate of individual endogenous EVs. We identified a subpopulation of EVs with exosome features, released in a syntenin-dependent manner from the yolk syncytial layer into the blood circulation. These exosomes are captured, endocytosed, and degraded by patrolling macrophages and endothelial cells in the caudal vein plexus (CVP) in a scavenger receptor- and dynamin-dependent manner. Interference with exosome biogenesis affected CVP growth, suggesting a role in trophic support. Altogether, our work represents a system for studying endogenous EV function in vivo with high spatiotemporal accuracy, demonstrating functional inter-organ communication by exosomes. (hide) EV-METRIC 57% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Dissociated embryo Sample origin Overexpression of CD63-phluorin in yolk syncitial layer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C IAF Adj. k-factor 41.45 (pelleting) / 41.45 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function, Biogenesis/cargo sorting, Mechanism of uptake/transfer, New methodological development, Identification of content (omics approaches), Interorgan transfer of EVs in vivo Sample Species Danio rerio Sample Type Dissociated embryo Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type TLA-120.1 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 41.45 Wash: time (min) 60 Wash: Rotor Type TLA-120.2 Wash: speed (g) 100000 Wash: adjusted k-factor 41.45 Immunoaffinity capture Selected surface protein(s) GFP Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Nanoparticle tracking analysis Extra particle analysis NTA Report type Modus Reported size (nm) 108 EV concentration Yes Particle yield 860000000000 EM EM-type Immune-EM EM protein GFP Image type Close-up, Wide-field Report size (nm) 60-200 Extra information We have developed live cell imaging method to visualize and quantify exosome release (Verweij et al., JCB 2018). This method could be added to EV-track, e.g. as a measure to positively identify the endosomal origin of an EV population.

	EV180078	10/10	Danio rerio	Zmel1	(d)(U)C	Hyenne V	2019	57%
Study summary Full title Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo. All authors Hyenne V, Ghoroghi S, Collot M, Bons J, Follain G, Harlepp S, Mary B, Bauer J, Mercier L, Busnelli I, Lefebvre O, Fekonja N, Garcia-Leon MJ, Machado P, Delalande F, López AA, Silva SG, Verweij FJ, van Niel G, Djouad F, Peinado H, Carapito C, Klymchenko AS, Goetz JG. Journal Dev cell Abstract Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly (show more...)Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: non-EV: Proteomics yes Show all info Study aim Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Danio rerio Sample Type Cell culture supernatant EV-producing cells Zmel1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 16 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 90

	EV210173	1/5	Homo sapiens	U87MG	(d)(U)C Filtration	Wang, Huayi	2019	56%
Study summary Full title Evaluation of serum extracellular vesicles as noninvasive diagnostic markers of glioma All authors Huayi Wang, Dengzhi Jiang, Wenzhe Li, Xiang Xiang, Jun Zhao, Bin Yu, Chen Wang, Zhaohui He, Ling Zhu, Yanlian Yang Journal Theranostics Abstract Rationale: Glioma is the most common malignant primary brain tumor in the central nervous system (CN (show more...)Rationale: Glioma is the most common malignant primary brain tumor in the central nervous system (CNS). The lack of reliable noninvasive diagnostic and prognostic methods is one of the main reasons for the high mortality of glioma. Serum has become a useful biomarker for the diagnosis and prognosis prediction of glioma because extracellular vesicles (EVs) carry molecular components from their parental cells. Methods: To detect EVs and perform molecular analysis of serum EVs, we established and optimized a microbead-assisted method based on flow cytometry and estimated the efficacy of EGFR protein expression and NLGN3 and PTTG1 mRNA in serum EVs from glioma patients (n=23) and healthy individuals (n=12). We evaluated the ability of EGFR+ EVs to differentiate high-grade and low-grade glioma patients and checked the correlation between EGFR in EVs and the ki-67 labeling index (LI) in the tumor tissue. Results: We demonstrated that EGFR+ EVs are effective diagnostic and prognostic markers of glioma. The expression of EGFR in serum EVs can accurately differentiate high-grade and low-grade glioma patients, and EGFR in EVs positively correlates with ki-67 LI in the tumor tissue. We also showed the potential of NLGN3 and PTTG1 mRNA in EVs for detecting glioma patients. Conclusions: We demonstrate that the protein expression of EGFR in serum EVs is an effective diagnostic marker of glioma. EGFR in EVs highly correlates with the malignancy of glioma. We also show the potential of NLGN3 and PTTG1 in EVs for detecting glioma. The optimized flow cytometry with the aid of microbead-based EV enrichment show its potential as a noninvasive method for the detection of glioma and will be beneficial to the management of glioma. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ Flotillin1/ EGFR non-EV: Grp94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87MG EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ EGFR/ CD81 Not detected contaminants Grp94 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins EGFR Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210173	2/5	Homo sapiens	U251	(d)(U)C Filtration	Wang, Huayi	2019	56%
Study summary Full title Evaluation of serum extracellular vesicles as noninvasive diagnostic markers of glioma All authors Huayi Wang, Dengzhi Jiang, Wenzhe Li, Xiang Xiang, Jun Zhao, Bin Yu, Chen Wang, Zhaohui He, Ling Zhu, Yanlian Yang Journal Theranostics Abstract Rationale: Glioma is the most common malignant primary brain tumor in the central nervous system (CN (show more...)Rationale: Glioma is the most common malignant primary brain tumor in the central nervous system (CNS). The lack of reliable noninvasive diagnostic and prognostic methods is one of the main reasons for the high mortality of glioma. Serum has become a useful biomarker for the diagnosis and prognosis prediction of glioma because extracellular vesicles (EVs) carry molecular components from their parental cells. Methods: To detect EVs and perform molecular analysis of serum EVs, we established and optimized a microbead-assisted method based on flow cytometry and estimated the efficacy of EGFR protein expression and NLGN3 and PTTG1 mRNA in serum EVs from glioma patients (n=23) and healthy individuals (n=12). We evaluated the ability of EGFR+ EVs to differentiate high-grade and low-grade glioma patients and checked the correlation between EGFR in EVs and the ki-67 labeling index (LI) in the tumor tissue. Results: We demonstrated that EGFR+ EVs are effective diagnostic and prognostic markers of glioma. The expression of EGFR in serum EVs can accurately differentiate high-grade and low-grade glioma patients, and EGFR in EVs positively correlates with ki-67 LI in the tumor tissue. We also showed the potential of NLGN3 and PTTG1 mRNA in EVs for detecting glioma patients. Conclusions: We demonstrate that the protein expression of EGFR in serum EVs is an effective diagnostic marker of glioma. EGFR in EVs highly correlates with the malignancy of glioma. We also show the potential of NLGN3 and PTTG1 in EVs for detecting glioma. The optimized flow cytometry with the aid of microbead-based EV enrichment show its potential as a noninvasive method for the detection of glioma and will be beneficial to the management of glioma. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ Flotillin1/ EGFR non-EV: Grp94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U251 EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ EGFR/ CD81 Not detected contaminants Grp94 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins EGFR Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210173	3/5	Homo sapiens	HA	(d)(U)C Filtration	Wang, Huayi	2019	56%
Study summary Full title Evaluation of serum extracellular vesicles as noninvasive diagnostic markers of glioma All authors Huayi Wang, Dengzhi Jiang, Wenzhe Li, Xiang Xiang, Jun Zhao, Bin Yu, Chen Wang, Zhaohui He, Ling Zhu, Yanlian Yang Journal Theranostics Abstract Rationale: Glioma is the most common malignant primary brain tumor in the central nervous system (CN (show more...)Rationale: Glioma is the most common malignant primary brain tumor in the central nervous system (CNS). The lack of reliable noninvasive diagnostic and prognostic methods is one of the main reasons for the high mortality of glioma. Serum has become a useful biomarker for the diagnosis and prognosis prediction of glioma because extracellular vesicles (EVs) carry molecular components from their parental cells. Methods: To detect EVs and perform molecular analysis of serum EVs, we established and optimized a microbead-assisted method based on flow cytometry and estimated the efficacy of EGFR protein expression and NLGN3 and PTTG1 mRNA in serum EVs from glioma patients (n=23) and healthy individuals (n=12). We evaluated the ability of EGFR+ EVs to differentiate high-grade and low-grade glioma patients and checked the correlation between EGFR in EVs and the ki-67 labeling index (LI) in the tumor tissue. Results: We demonstrated that EGFR+ EVs are effective diagnostic and prognostic markers of glioma. The expression of EGFR in serum EVs can accurately differentiate high-grade and low-grade glioma patients, and EGFR in EVs positively correlates with ki-67 LI in the tumor tissue. We also showed the potential of NLGN3 and PTTG1 mRNA in EVs for detecting glioma patients. Conclusions: We demonstrate that the protein expression of EGFR in serum EVs is an effective diagnostic marker of glioma. EGFR in EVs highly correlates with the malignancy of glioma. We also show the potential of NLGN3 and PTTG1 in EVs for detecting glioma. The optimized flow cytometry with the aid of microbead-based EV enrichment show its potential as a noninvasive method for the detection of glioma and will be beneficial to the management of glioma. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ Flotillin1/ EGFR non-EV: GRP94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HA EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD81 Not detected EV-associated proteins EGFR Not detected contaminants GRP94 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins EGFR Characterization: Lipid analysis No Characterization: Particle analysis None

	EV190080	1/3	Homo sapiens	OVCAR5	DG (d)(U)C SEC SEC (non-commercial)	Zaborowski MP	2019	56%
Study summary Full title Effects of high-dose phytoestrogens on circulating cellular microparticles and coagulation function in postmenopausal women. All authors Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, Zappulli V, Maas SLN, Allen RM, Rumde P, György B, Aufiero M, Schweiger MW, Lai CP, Weissleder R, Lee H, Vickers KC, Tannous BA, Breakefield XO. Journal Sci Rep Abstract Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter (show more...)Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C SEC SEC (non-commercial) Protein markers EV: CD63 non-EV: Proteomics no EV density (g/ml) 1.11 Show all info Study aim New methodological development/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells OVCAR5 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120101 Wash: volume per pellet (ml) 40 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 120101 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 8% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.5 Orientation Top-down Rotor type MLS-50 Speed (g) 200620 Duration (min) 38 Fraction volume (mL) 350 Fraction processing None Size-exclusion chromatography Total column volume (mL) 24 Sample volume/column (mL) 1 Resin type Superdex 200 Other Name other separation method SEC (non-commercial) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Other 1 https://www.ncbi.nlm.nih.gov/pubmed/30943406 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-120 Other particle analysis name(1) Report type EV-concentration

	EV190049	1/3	Homo sapiens	Expi293F	(d)(U)C qEV	Bliss CM	2019	56%
Study summary Full title Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice. All authors Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L. Journal Matters Abstract Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV Protein markers EV: CD81/ Alix/ CD63/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Antigen targeting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported

	EV190049	2/3	Homo sapiens	Expi293F	(d)(U)C qEV	Bliss CM	2019	56%
Study summary Full title Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice. All authors Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L. Journal Matters Abstract Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Transfected with plasmid expressing EGFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV Protein markers EV: CD81/ Alix/ CD63/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Antigen targeting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 161 EV concentration Yes

	EV190049	3/3	Homo sapiens	Expi293F	(d)(U)C qEV	Bliss CM	2019	56%
Study summary Full title Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice. All authors Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L. Journal Matters Abstract Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Transfected with plasmid expressing EGFP_C1C2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV Protein markers EV: CD81/ Alix/ CD63/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Antigen targeting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 157 EV concentration Yes

	EV190027	1/1	Mus musculus	primary oligodendrocytes	(d)(U)C	Auber M	2019	56%
Study summary Full title Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles. All authors Auber M, Fröhlich D, Drechsel O, Karaulanov E, Krämer-Albers EM. Journal J Extracell Vesicles Abstract Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from (show more...)Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from cultured cells may co-purify RNAs derived from media supplements such as fetal bovine serum (FBS) confounding EV-associated RNA. Defined culture media supplemented with a range of nutrient components provide an alternative to FBS addition and allow EV-collection under full medium conditions avoiding starvation and cell stress during the collection period. However, the potential contribution of serum-free media supplements to EV-RNA contamination has remained elusive and has never been assessed. Here, we report that RNA isolated from EVs harvested from cells under serum-replacement conditions includes miRNA contaminants carried into the sample by defined media components. Subjecting unconditioned, EV-free medium to differential centrifugation followed by reverse transcription quantitative PCR (RT-qPCR) on RNA isolated from the pellet resulted in detection of miRNAs that had been classified as EV-enriched by RNA-seq or RT-qPCR of an isolated EV-fraction. Ribonuclease (RNase-A) and detergent treatment removed most but not all of the contaminating miRNAs. Further analysis of the defined media constituents identified Catalase as a main source of miRNAs co-isolating together with EVs. Hence, miRNA contaminants can be carried into EV-samples even under serum-free harvesting conditions using culture media that are expected to be chemically defined. Formulation of miRNA-free media supplements may provide a solution to collect EVs clean from confounding miRNAs, which however still remains a challenging task. Differential analysis of EVs collected under full medium and supplement-deprived conditions appears to provide a strategy to discriminate confounding and EV-associated RNA. In conclusion, we recommend careful re-evaluation and validation of EV small RNA-seq and RT-qPCR datasets by determining potential medium background. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: see EV130009 non-EV: see EV130009 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary oligodendrocytes EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 103000 Characterization: Protein analysis PMID previous EV protein analysis see EV130009 Protein Concentration Method Not determined Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis

	EV190026	1/2	Homo sapiens	Serum	(d)(U)C qEV	Gualerzi A	2019	56%
Study summary Full title Raman profiling of circulating extracellular vesicles for the stratification of Parkinson's patients. All authors Gualerzi A, Picciolini S, Carlomagno C, Terenzi F, Ramat S, Sorbi S, Bedoni M. Journal Nanomedicine Abstract Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clin (show more...)Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clinical heterogeneity. Extracellular vesicles (EVs) were proposed as new biomarkers for PD because of their role as vehicles of multiple PD related molecules, but technical limitations exist in their detection and characterization in a clinical environment. We propose herein a Raman based protocol for the label-free analysis of circulating EVs as diagnostic and predictive tool for PD. After purification from serum of PD patients and healthy subjects, EVs were analyzed by Raman spectroscopy demonstrating the feasibility and reproducibility of the proposed biophotonic approach, its moderate accuracy in distinguishing PD patients from controls by their EV profile and the correlation between Raman data and clinical scales. Once validated, the Raman spectroscopy of circulating EVs could represent a reliable, automatable and sensitive method for the stratification of PD patients and for the evaluation of the effectiveness of rehabilitation and pharmacological treatments. (hide) EV-METRIC 56% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV Protein markers EV: CD63/ Flotillin1/ alpha-synuclein non-EV: Albumin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD63/ alpha-synuclein Detected contaminants Albumin Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

	EV190026	2/2	Homo sapiens	Serum	(d)(U)C qEV	Gualerzi A	2019	56%
Study summary Full title Raman profiling of circulating extracellular vesicles for the stratification of Parkinson's patients. All authors Gualerzi A, Picciolini S, Carlomagno C, Terenzi F, Ramat S, Sorbi S, Bedoni M. Journal Nanomedicine Abstract Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clin (show more...)Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clinical heterogeneity. Extracellular vesicles (EVs) were proposed as new biomarkers for PD because of their role as vehicles of multiple PD related molecules, but technical limitations exist in their detection and characterization in a clinical environment. We propose herein a Raman based protocol for the label-free analysis of circulating EVs as diagnostic and predictive tool for PD. After purification from serum of PD patients and healthy subjects, EVs were analyzed by Raman spectroscopy demonstrating the feasibility and reproducibility of the proposed biophotonic approach, its moderate accuracy in distinguishing PD patients from controls by their EV profile and the correlation between Raman data and clinical scales. Once validated, the Raman spectroscopy of circulating EVs could represent a reliable, automatable and sensitive method for the stratification of PD patients and for the evaluation of the effectiveness of rehabilitation and pharmacological treatments. (hide) EV-METRIC 56% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Parkinson's disease Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV Protein markers EV: CD63/ Flotillin1/ alpha-synuclein non-EV: Albumin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD63/ alpha-synuclein Detected contaminants Albumin Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up

	EV180078	6/10	Mus musculus	4T1	(d)(U)C DG	Hyenne V	2019	56%
Study summary Full title Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo. All authors Hyenne V, Ghoroghi S, Collot M, Bons J, Follain G, Harlepp S, Mary B, Bauer J, Mercier L, Busnelli I, Lefebvre O, Fekonja N, Garcia-Leon MJ, Machado P, Delalande F, López AA, Silva SG, Verweij FJ, van Niel G, Djouad F, Peinado H, Carapito C, Klymchenko AS, Goetz JG. Journal Dev cell Abstract Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly (show more...)Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ TSG101 non-EV: Proteomics yes EV density (g/ml) 1.14 Show all info Study aim Function/New methodological development/Identification of content (omics approaches)/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 dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 16 Wash: time (min) 70 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16 Sample volume (mL) 1 Orientation Top-down Rotor type SW 28 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 3 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported

	EV200141	1/2	Homo sapiens	Blood plasma	Exoquick	Zhang, Weiting	2019	50%
Study summary Full title Detection of fetal trisomy and single gene disease by massively parallel sequencing of extracellular vesicle DNA in maternal plasma: a proof-of-concept validation All authors Weiting Zhang, Sen Lu, Dandan Pu, Haiping Zhang, Lin Yang, Peng Zeng, Fengxia Su, Zhichao Chen, Mei Guo, Ying Gu, Yanmei Luo, Huamei Hu, Yanping Lu, Fang Chen, Ya Gao Journal BMC Genomics Abstract Background: During human pregnancy, placental trophectoderm cells release extracellular vesicles (EV (show more...)Background: During human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases. Methods: Maternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data. Results: EVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations. Conclusions: We proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Healthy pregnant Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Exoquick Protein markers EV: CD81/ PLAP/ CD63/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Exoquick Other Name other separation method Exoquick 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/ PLAP/ CD81 Not detected contaminants Calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD9 Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-50nm

	EV200048	1/2	Gallus gallus	semen	IAF (d)(U)C ExoQuick Filtration	Sheng Chen	2019	50%
Study summary Full title MicroRNA expression profile in extracellular vesicles derived from ALV-J infected chicken semen All authors Sheng Chen, Liqin Liao, Qiqi Zhao, Xinheng Zhang, Hongxin Li, Wencheng Lin, Feng Chen, Qingmei Xie Journal Virus Res Abstract MicroRNAs(miRNAs) have been reported to regulate gene expression in many processes. MiRNA in extrace (show more...)MicroRNAs(miRNAs) have been reported to regulate gene expression in many processes. MiRNA in extracellular vesicles (EVs) also have been widely investigated, while there is no studies of miRNAs in seminal EVs. Subgroup J of Avian leukosis virus (ALV-J) can be transmitted vertically, but the mechanism of it is not clear enough. This study was to examine the miRNA expression profile in seminal EVs and inquire into the relation between it and the vertical transmission by performing gene ontology (GO) and pathway enrichment analysis. Here, we first isolated and characterized seminal EVs by Nanoparticle Tracking Analysis、Western Blot and Transmission electron microscopy experiments. By deep sequencing of each EVs miRNA library, 9 typical differentially expressed miRNA, including 6 up-regulated and 3 down-regulated, were identified. Gene target prediction, GO annotation and KEGG pathway enrichment analysis showed possible function associated with these miRNAs. Overall, these findings will increase our understanding of the content and composition of miRNA in seminal EVs and provide new insights into the important role of the seminal EVs miRNAs regulation in ALV-J transmission. (hide) EV-METRIC 50% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type semen 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) (d)(U)C Commercial method Filtration Protein markers EV: CD81/ HSP70/ TSG101/ CD63/ CD9 non-EV: GRP78 Proteomics no Show all info Study aim NA Sample Species Gallus gallus Sample Type semen Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ HSP70/ CD81 Not detected EV-associated proteins HSP70/ CD81/ TSG101/ CD63/ CD9 Not detected contaminants GRP78 ELISA Antibody details provided? No Detected EV-associated proteins HSP70/ CD63/ CD81/ CD9/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 110 EV concentration Yes Particle yield particles/ml;Yes, other: 858000000

	EV200045	3/5	Homo sapiens	PCI-13	(d)(U)C SEC (non-commercial) Filtration UF	Ludwig Nils	2019	50%
Study summary Full title Isolation and Analysis of Tumor-Derived Exosomes All authors Ludwig N, Hong CS, Ludwig S, Azambuja JH, Sharma P, Theodoraki MN, Whiteside TL. Journal Current Protocols in Immunology Abstract A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presen (show more...)A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presented. Tumor-derived exosomes (TEX) are defined as a subset of extracellular vesicles (EVs) sized at 30 to 150 nm and originating from multivesicular bodies (MVBs). The method utilizes size exclusion chromatography (SEC) for recovery of exosomes from cell-line supernatants or cancer patients' plasma. The recovered exosomes are morphologically intact, aggregate-free, and functionally competent. Their molecular content parallels that of the parent tumor cells and they carry various immunoregulatory ligands known to modulate functions of immune cells. All exosomes isolated from tumor cell lines are TEX, while those isolated from plasma of cancer patients have to be fractionated into TEX and non-TEX. Mini-SEC allows for exosome isolation and recovery in quantities sufficient for molecular profiling, functional studies, and, in the case of plasma, further fractionation into TEX and non-TEX. The mini-SEC method can also be used for comparative studies of the exosome content in serial specimens of cancer patients' body fluids. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C SEC (non-commercial) Filtration UF Protein markers EV: None non-EV: Proteomics no Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PCI-13 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method SEC (non-commercial) PMID previous EV protein analysis PMID 30042174 + PMID 30370252 Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins PMID 30042174 / PMID 30370252 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Not Reported EM EM-type Transmission-EM Image type Wide-field

	EV190040	3/12	Homo sapiens	HEK293T	DG UF	Geeurickx E	2019	50%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin gag-EGFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: non-EV: Proteomics yes EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium Cell viability (%) 96 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 Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Used subtypes 1.046 1.068 g/ml Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database Yes: Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV190040	8/12	Homo sapiens	primary fibroblasts	DG UF	Geeurickx E	2019	50%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: non-EV: Proteomics yes EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary fibroblasts EV-harvesting Medium Serum free medium Cell viability (%) 96 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 Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Proteomics database Yes: Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV190025	1/4	Homo sapiens	Blood plasma	(d)(U)C Filtration qEV	Czystowska-Kuzmicz, Malgorzata	2019	50%
Study summary Full title Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma All authors Malgorzata Czystowska-Kuzmicz, Anna Sosnowska, Dominika Nowis, Kavita Ramji, Marta Szajnik, Justyna Chlebowska-Tuz, Ewa Wolinska, Pawel Gaj, Magdalena Grazul, Zofia Pilch, Abdessamad Zerrouqi, Agnieszka Graczyk-Jarzynka, Karolina Soroczynska, Szczepan Cierniak, Robert Koktysz, Esther Elishaev, Slawomir Gruca, Artur Stefanowicz, Roman Blaszczyk, Bartlomiej Borek, Anna Gzik, Theresa Whiteside, and Jakub Golab Journal Nat Commun Abstract Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (show more...)Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 (d)(U)C Filtration qEV Protein markers EV: TSG101/ ARG1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function 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 Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ ARG1/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 128 EV concentration Yes TRPS Report type Mean Reported size (nm) 65 EV concentration Yes

	EV190025	2/4	Homo sapiens	Blood plasma	(d)(U)C Filtration qEV	Czystowska-Kuzmicz, Malgorzata	2019	50%
Study summary Full title Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma All authors Malgorzata Czystowska-Kuzmicz, Anna Sosnowska, Dominika Nowis, Kavita Ramji, Marta Szajnik, Justyna Chlebowska-Tuz, Ewa Wolinska, Pawel Gaj, Magdalena Grazul, Zofia Pilch, Abdessamad Zerrouqi, Agnieszka Graczyk-Jarzynka, Karolina Soroczynska, Szczepan Cierniak, Robert Koktysz, Esther Elishaev, Slawomir Gruca, Artur Stefanowicz, Roman Blaszczyk, Bartlomiej Borek, Anna Gzik, Theresa Whiteside, and Jakub Golab Journal Nat Commun Abstract Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (show more...)Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin ovarian cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration qEV Protein markers EV: TSG101/ ARG1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function 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 Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ ARG1/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 126 EV concentration Yes TRPS Report type Mean Reported size (nm) 58 EV concentration Yes

	EV190021	2/4	Homo sapiens	Blood plasma	(d)(U)C UF	Yunusova, Natalia	2019	50%
Study summary Full title Metalloproteinases at the surface of small extrcellular vesicles in advanced ovarian cancer: Relationships with ascites volume and peritoneal canceromatosis index All authors Natalia V Yunusova, Marina R Patysheva, Sergey V Molchanov, Elena A Zambalova, Alina E Grigor'eva, Larisa A Kolomiets, Maxim O Ochirov, Svetlana N Tamkovich, Irina V Kondakova Journal Clinica Chimica Acta Abstract Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essent (show more...)Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essential role in the regulation of signaling from growth factors receptors and adhesion molecules, cell motility and extracellular matrix degradation. The aim of the study was to evaluate the relationship between the levels of small extracellular vesicles (sEVs) metalloproteinases, such as ADAM10, ADAM17, MMP2, MMP9 and EMMPRIN and ascites volume and peritoneal canceromatosis index in advanced ovarian cancer patients (OCPs). The subpopulations of metalloproteinases at the surface of sEVs of borderline ovarian tumor patients (BOTPs) (n = 20, 36.5 ± 2.5 years) and previously untreated advanced OCPs (n = 35, 56.5 ± 2.5 years) were evaluated using flow cytometry. The metalloproteinase subpopulations of CD9-positive sEVs isolated from plasma of BOTPs and OCPs appeared to be quite similar. However, a significant difference in the expression of ADAM-metalloproteinases in ascites sEVs was found between BOTPs and OCPs. The level of sEVs metalloproteinases in OCPs significantly depended on the ascites volume. A statistically significant relationship between the level of ADAM10+/ADAM17- subpopulation in plasma sEVs and the peritoneal canceromatosis index was found (R = 0.66, p < .05). The levels of metalloproteinases and EMMPRIN in circulating sEVs, as well as the assessment of individual subpopulations may be promising approaches to OCPs managing. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin advanced ovarian cancer, pretreatment Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: / CD81/ CD63/ CD9/ CD24 non-EV: / Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 16 Wash: time (min) 90 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD24/ CD9/ CD63/ CD81 Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV190021	3/4	Homo sapiens	ascites	(d)(U)C UF	Yunusova, Natalia	2019	50%
Study summary Full title Metalloproteinases at the surface of small extrcellular vesicles in advanced ovarian cancer: Relationships with ascites volume and peritoneal canceromatosis index All authors Natalia V Yunusova, Marina R Patysheva, Sergey V Molchanov, Elena A Zambalova, Alina E Grigor'eva, Larisa A Kolomiets, Maxim O Ochirov, Svetlana N Tamkovich, Irina V Kondakova Journal Clinica Chimica Acta Abstract Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essent (show more...)Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essential role in the regulation of signaling from growth factors receptors and adhesion molecules, cell motility and extracellular matrix degradation. The aim of the study was to evaluate the relationship between the levels of small extracellular vesicles (sEVs) metalloproteinases, such as ADAM10, ADAM17, MMP2, MMP9 and EMMPRIN and ascites volume and peritoneal canceromatosis index in advanced ovarian cancer patients (OCPs). The subpopulations of metalloproteinases at the surface of sEVs of borderline ovarian tumor patients (BOTPs) (n = 20, 36.5 ± 2.5 years) and previously untreated advanced OCPs (n = 35, 56.5 ± 2.5 years) were evaluated using flow cytometry. The metalloproteinase subpopulations of CD9-positive sEVs isolated from plasma of BOTPs and OCPs appeared to be quite similar. However, a significant difference in the expression of ADAM-metalloproteinases in ascites sEVs was found between BOTPs and OCPs. The level of sEVs metalloproteinases in OCPs significantly depended on the ascites volume. A statistically significant relationship between the level of ADAM10+/ADAM17- subpopulation in plasma sEVs and the peritoneal canceromatosis index was found (R = 0.66, p < .05). The levels of metalloproteinases and EMMPRIN in circulating sEVs, as well as the assessment of individual subpopulations may be promising approaches to OCPs managing. (hide) EV-METRIC 50% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type ascites Sample origin borderline ovarian tumors, control pts Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: / CD81/ CD63/ CD9/ CD24 non-EV: / Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 16 Wash: time (min) 90 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD24/ CD9/ CD81/ CD63 Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV190021	4/4	Homo sapiens	ascites	(d)(U)C UF	Yunusova, Natalia	2019	50%
Study summary Full title Metalloproteinases at the surface of small extrcellular vesicles in advanced ovarian cancer: Relationships with ascites volume and peritoneal canceromatosis index All authors Natalia V Yunusova, Marina R Patysheva, Sergey V Molchanov, Elena A Zambalova, Alina E Grigor'eva, Larisa A Kolomiets, Maxim O Ochirov, Svetlana N Tamkovich, Irina V Kondakova Journal Clinica Chimica Acta Abstract Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essent (show more...)Metalloproteinases and their extracellular matrix metalloproteinase inducer (EMMPRIN) play an essential role in the regulation of signaling from growth factors receptors and adhesion molecules, cell motility and extracellular matrix degradation. The aim of the study was to evaluate the relationship between the levels of small extracellular vesicles (sEVs) metalloproteinases, such as ADAM10, ADAM17, MMP2, MMP9 and EMMPRIN and ascites volume and peritoneal canceromatosis index in advanced ovarian cancer patients (OCPs). The subpopulations of metalloproteinases at the surface of sEVs of borderline ovarian tumor patients (BOTPs) (n = 20, 36.5 ± 2.5 years) and previously untreated advanced OCPs (n = 35, 56.5 ± 2.5 years) were evaluated using flow cytometry. The metalloproteinase subpopulations of CD9-positive sEVs isolated from plasma of BOTPs and OCPs appeared to be quite similar. However, a significant difference in the expression of ADAM-metalloproteinases in ascites sEVs was found between BOTPs and OCPs. The level of sEVs metalloproteinases in OCPs significantly depended on the ascites volume. A statistically significant relationship between the level of ADAM10+/ADAM17- subpopulation in plasma sEVs and the peritoneal canceromatosis index was found (R = 0.66, p < .05). The levels of metalloproteinases and EMMPRIN in circulating sEVs, as well as the assessment of individual subpopulations may be promising approaches to OCPs managing. (hide) EV-METRIC 50% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type ascites Sample origin advanced ovarian cancer, pretreatment Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: / CD81/ CD63/ CD9/ CD24 non-EV: / Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 16 Wash: time (min) 90 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD24/ CD9/ CD63/ CD81 Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV190003	1/1	Homo sapiens	Urine	(d)(U)C	Sabaratnam R	2019	50%
Study summary Full title In human nephrectomy specimens, the kidney level of tubular transport proteins does not correlate with their abundance in urinary extracellular vesicles. All authors Sabaratnam R, Geertsen L, Skjødt K, Hojlund K, Dimke H, Lund L, Svenningsen P. Journal Am J Physiol Renal Physiol Abstract Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumptio (show more...)Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumption for years has been that uEVs might provide a non-invasive liquid biopsy that reflect physiological regulation of transporter protein expression in human. We hypothesized that protein abundance in human kidney tissue and uEV are directly related and tested this in paired collections of nephrectomy tissue and urine sample from 12 patients. Kidney tissue was fractioned into total kidney protein, crude membrane (plasma membrane and large intracellular vesicles) and intracellular vesicle enriched fractions, as well as sections for immunolabelling. uEVs were isolated from spot urine samples. Antibodies were used to quantify 6 segment-specific proteins (proximal tubular expressed Na/Phosphate cotransporter NaPi-2a, thick ascending limb expressed Tamm-Horsfall protein and renal-outer-medullary K+channel ROMK, distal convoluted tubular expressed NaCl cotransporter NCC, intercalated cell expressed proton-pump subunit ATP6V1G3 and principal cell expressed aquaporin 2 (AQP2)) and 3 uEV markers (exosomal CD63, microvesicle marker VAMP3 and β-actin) in each fractions. By western blotting and immunofluorescence labelling, we found significant positive correlations between abundance of CD63, NCC, AQP2 and ATP6V1G3, respectively, within the different kidney-derived fractions. We detected all 9 proteins in uEVs, but their level did not correlate with kidney tissue protein abundance. The uEV protein levels showed higher inter-patient variability than the kidney-derived fractions, indicating that factors, besides kidney protein abundance, contribute to the uEV protein level. Our data suggest that, in a random sample of nephrectomy patients, uEV protein level is not a predictor of kidney protein abundance. (hide) EV-METRIC 50% (86th 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 pre-nephrectomy Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: SLC34A1/ VAMP3/ CD63/ beta-actin/ ROMK non-EV: Tamm-Horsfall protein 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 Pelleting performed No Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ VAMP3/ beta-actin/ SLC34A1/ ROMK Detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No

	EV180071	2/3	Homo sapiens	Blood plasma	SEC UF	Brahmer A	2019	50%
Study summary Full title Platelets, endothelial cells and leukocytes contribute to the exercise-triggered release of extracellular vesicles into the circulation. All authors Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM. Journal J Extracell Vesicles Abstract Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin RQ 0.9 during exercise 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 SEC UF Protein markers EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142 non-EV: / ApoA1 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101/ CD9/ Syntenin/ CD41b/ CD63 Not detected EV-associated proteins Sarcoglycan-alpha Detected contaminants ApoA1 Not detected contaminants Detected EV-associated proteins CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported

	EV180071	3/3	Homo sapiens	Blood plasma	SEC UF	Brahmer A	2019	50%
Study summary Full title Platelets, endothelial cells and leukocytes contribute to the exercise-triggered release of extracellular vesicles into the circulation. All authors Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM. Journal J Extracell Vesicles Abstract Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin post exercise 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 SEC UF Protein markers EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142 non-EV: / ApoA1 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101/ CD9/ Syntenin/ CD41b/ CD63 Not detected EV-associated proteins Sarcoglycan-alpha Detected contaminants ApoA1 Not detected contaminants Detected EV-associated proteins CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported

	EV210242	1/2	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Jayabalan N	2019	45%
Study summary Full title Quantitative Proteomics by SWATH-MS Suggest an Association Between Circulating Exosomes and Maternal Metabolic Changes in Gestational Diabetes Mellitus. All authors Jayabalan N, Lai A, Nair S, Guanzon D, Scholz-Romero K, Palma C, McIntyre HD, Lappas M, Salomon C Journal Proteomics Abstract Several factors including placental hormones (PH) released from the human placenta have been associa (show more...)Several factors including placental hormones (PH) released from the human placenta have been associated with the development of insulin resistance and gestational diabetes mellitus (GDM). However, circulating levels of PH does not correlate well with maternal insulin sensitivity across gestation, suggesting that other, previously unrecognized, mechanisms may be involved. The levels of circulating exosomes are higher in GDM compared to normal. GDM derived exosomes produce greater release of pro-inflammatory cytokines from endothelial cells compared to exosomes from normal, suggesting that their contents may differ compared to normal pregnancies. Using a quantitative, information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]) approach, differentially abundant circulating exosome proteins are identified in women with normal glucose tolerance (NGT) and GDM at the time of GDM diagnosis. A total of 78 statistically significant proteins in the relative expression of exosomal proteins in GDM are compared with NGT. Bioinformatic analysis shows that the exosomal proteins in GDM target pathways are mainly associated with energy production, inflammation, and metabolism. Finally, an independent cohort of patients is used to validate some of the proteins identified by SWATH. The data obtained may be of utility in elucidating the underlying physiological mechanisms associated with insulin resistance in GDM. (hide) EV-METRIC 45% (79th 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 Healthy pregnant Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD9/ TSG101/ CD63 non-EV: Grp94 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ CD63 Detected contaminants Grp94 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 105 +/- 15nm EV concentration Yes Particle yield No: 0.00e+0

	EV210242	2/2	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Jayabalan N	2019	45%
Study summary Full title Quantitative Proteomics by SWATH-MS Suggest an Association Between Circulating Exosomes and Maternal Metabolic Changes in Gestational Diabetes Mellitus. All authors Jayabalan N, Lai A, Nair S, Guanzon D, Scholz-Romero K, Palma C, McIntyre HD, Lappas M, Salomon C Journal Proteomics Abstract Several factors including placental hormones (PH) released from the human placenta have been associa (show more...)Several factors including placental hormones (PH) released from the human placenta have been associated with the development of insulin resistance and gestational diabetes mellitus (GDM). However, circulating levels of PH does not correlate well with maternal insulin sensitivity across gestation, suggesting that other, previously unrecognized, mechanisms may be involved. The levels of circulating exosomes are higher in GDM compared to normal. GDM derived exosomes produce greater release of pro-inflammatory cytokines from endothelial cells compared to exosomes from normal, suggesting that their contents may differ compared to normal pregnancies. Using a quantitative, information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]) approach, differentially abundant circulating exosome proteins are identified in women with normal glucose tolerance (NGT) and GDM at the time of GDM diagnosis. A total of 78 statistically significant proteins in the relative expression of exosomal proteins in GDM are compared with NGT. Bioinformatic analysis shows that the exosomal proteins in GDM target pathways are mainly associated with energy production, inflammation, and metabolism. Finally, an independent cohort of patients is used to validate some of the proteins identified by SWATH. The data obtained may be of utility in elucidating the underlying physiological mechanisms associated with insulin resistance in GDM. (hide) EV-METRIC 45% (79th 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 Pregnant with gestational diabetes mellitus Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD9/ TSG101/ CD63 non-EV: Grp94 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ CD63 Detected contaminants Grp94 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 102 +/- 17nm EV concentration Yes Particle yield No: 0.00e+0

	EV200143	1/2	Homo sapiens	Blood plasma	(d)(U)C Filtration	Ramkumar, Menon	2019	45%
Study summary Full title Circulating Exosomal miRNA Profile During Term and Preterm Birth Pregnancies: A Longitudinal Study All authors Ramkumar Menon, Chirantan Debnath, Andrew Lai, Dominic Guanzon, Shinjini Bhatnagar, Pallavi K Kshetrapal, Samantha Sheller-Miller, Carlos Salomon, Garbhini Study Team Journal Endocrinology Abstract Despite decades of research in the field of human reproduction, the mechanisms responsible for human (show more...)Despite decades of research in the field of human reproduction, the mechanisms responsible for human parturition still remain elusive. The objective of this study was to describe the changes in the exosomal miRNA concentrations circulating in the maternal plasma between mothers delivering term and preterm neonates, across gestation using a longitudinal study design. This descriptive study identifies the miRNA content in exosomes present in maternal plasma of term and preterm birth (PTB) (n = 20 and n = 10 per each gestational period, respectively) across gestation (i.e., first, second, and third trimesters and at the time of delivery). Changes in exosomal miRNA signature in maternal plasma during term and preterm gestation were determined using the NextSeq 500 high-output 75 cycles sequencing platform. A total of 167 and 153 miRNAs were found to significantly change (P < 0.05) as a function of the gestational age across term and PTB pregnancies, respectively. Interestingly, a comparison analysis between the exosomal miRNA profile between term and PTB reveals a total of 173 miRNAs that significantly change (P < 0.05) across gestation. Specific trends of changes (i.e., increase, decrease, and both) as a function of the gestational age were also identified. The bioinformatics analyses establish that the differences in the miRNA profile are targeting signaling pathways associated with TGF-β signaling, p53, and glucocorticoid receptor signaling, respectively. These data suggest that the miRNA content of circulating exosomes in maternal blood might represent a biomolecular fingerprint of the progression of pregnancy. (hide) EV-METRIC 45% (79th 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 Normal pregnancy Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD63/ CD9 non-EV: Grp94 Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected contaminants Grp94 Characterization: RNA analysis RNA analysis Type RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-120nm EM EM-type Transmission-EM Image type Close-up

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