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Results list Most used separation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV200179	3/4	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration DG UF	Ouyang, Yingshi	2016	88%
Study summary Full title Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity All authors Yingshi Ouyang, Avraham Bayer, Tianjiao Chu, Vladimir A Tyurin, Valerian E Kagan, Adrian E Morelli, Carolyn B Coyne, Yoel Sadovsky Journal Placenta Abstract Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among (show more...)Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. (hide) EV-METRIC 88% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name Primary human trophoblast 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Density gradient Ultrafiltration Protein markers EV: TSG101/ CD63/ Syntenin-1 non-EV: Argonaute2 Proteomics yes EV density (g/ml) 1.08-1.10 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Primary human trophoblast EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight, 100000g or commercial Separation Method Differential ultracentrifugation centrifugation steps Between 100,000 g and 150,000 g Density gradient Density medium Iodixanol Type Continuous Lowest density fraction 6 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type Not specified Speed (g) 100000 Duration (min) Overnight Fraction volume (mL) . Fraction processing Ultrafiltration Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 kda Membrane type PES Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ Syntenin-1/ TSG101 Not detected contaminants Argonaute2 Flow cytometry Hardware adjustments Proteomics Proteomics database Yes: Data and Specimen Hub Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mode Reported size (nm) 80-90nm EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV160001	1/1	Mus musculus	Cell culture supernatant	DG (d)(U)C	Stremersch S	2016	87%
Study summary Full title Comparing exosome-like vesicles with liposomes for the functional cellular delivery of small RNAs. All authors Stremersch S, Vandenbroucke RE, Van Wonterghem E, Hendrix A, De Smedt SC, Raemdonck K Journal J Control Release Abstract Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natu (show more...)Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natural carriers for biomolecule transfer between cells. This unique feature rationalizes their exploitation as bio-inspired drug delivery systems. However, the therapeutic application of ELVs is hampered by the lack of efficient and reproducible drug loading methods, in particular for therapeutic macromolecules. To overcome this limitation, we present a generic method to attach siRNA to the surface of isolated ELVs by means of a cholesterol anchor. Despite a feasible uptake in both a dendritic and lung epithelial cell line, B16F10- and JAWSII-derived ELVs were unable to functionally deliver the associated small RNAs, neither exogenous cholesterol-conjugated siRNA nor endogenous miRNA derived from the melanoma producer cell. The latter results were confirmed both for purified ELVs and ELVs delivered via a transwell co-culture set-up. In contrast, simple anionic fusogenic liposomes were able to induce a marked siRNA-mediated gene knockdown under equal experimental conditions, both indicating successful cytosolic delivery of surface-bound cholesterol-conjugated siRNA and further underscoring the incapacity of the here evaluated ELVs to guide cytosolic delivery of small RNAs. In conclusion, we demonstrate that a more in-depth understanding of the biomolecular delivery mechanism and specificity is required before ELVs can be envisioned as a generic siRNA carrier. (hide) EV-METRIC 87% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name B16F10, JAWSII Sample origin Control condition Focus vesicles exosome-like vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD81/ HSP70/ beta-actin/ CD63 non-EV: GM130/ Calreticulin Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells B16F10, JAWSII EV-harvesting Medium EV-depleted serum Preparation of EDS Ultrafiltration (MWCO 300 kDa) Cell viability 95 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Density gradient Only used for validation of main results Yes Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 0.125 Highest density fraction 0.5 Sample volume (mL) 1 Orientation Top-down (sample migrates downwards) Rotor type SW 55 Ti Speed (g) 200000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 150 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 115.5 Pelleting-wash: volume per pellet (mL) 5 Pelleting-wash: duration (min) 70 Pelleting-wash: rotor type 115.5 Pelleting-wash: speed (g) SW 55 Ti Pelleting-wash: adjusted k-factor 115.5 EV-subtype Used subtypes NO Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, HSP70, beta-actin Flow cytometry specific beads Selected surface protein(s) CD63 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-300 EV concentration Yes Particle yield 1000 EM EM-type Cryo-EM Image type Close-up

	EV210135	1/2	Homo sapiens	Cell culture supernatant	DG (d)(U)C UF	Stremersch, Stephan	2016	78%
Study summary Full title Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy All authors Stephan Stremersch, Monica Marro, Bat-El Pinchasik, Pieter Baatsen, An Hendrix, Stefaan C De Smedt, Pablo Loza-Alvarez, Andre G Skirtach, Koen Raemdonck, Kevin Braeckmans Journal Small Abstract Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention f (show more...)Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name Red blood cells 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C UF Protein markers EV: CD81/ HSP70/ CD63/ ?-actin non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Red blood cells EV-harvesting Medium Serum free medium Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 12.5% Highest density fraction 50% Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Top-down Rotor type SW 55 Ti Speed (g) 150000 Duration (min) 900 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 150 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 150000 Ultra filtration Cut-off size (kDa) 30 Membrane type Not specified Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ ?-actin/ HSP70/ CD81 Flow cytometry Hardware adjustments Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 170 Particle yield Not reported EM EM-type Cryo-EM Image type Wide-field EV concentration Not

	EV210135	2/2	Mus musculus	Cell culture supernatant	DG (d)(U)C UF	Stremersch, Stephan	2016	78%
Study summary Full title Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy All authors Stephan Stremersch, Monica Marro, Bat-El Pinchasik, Pieter Baatsen, An Hendrix, Stefaan C De Smedt, Pablo Loza-Alvarez, Andre G Skirtach, Koen Raemdonck, Kevin Braeckmans Journal Small Abstract Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention f (show more...)Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name B16F10 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C UF Protein markers EV: CD81/ HSP70/ CD63/ ?-actin non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods/New methodological development Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells B16F10 EV-harvesting Medium EV-depleted medium Preparation of EDS Other preparation;Ultrafiltration Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 12.5% Highest density fraction 50% Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Top-down Rotor type SW 55 Ti Speed (g) 150000 Duration (min) 900 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 150 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 150000 Ultra filtration Cut-off size (kDa) 30 Membrane type Not specified Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ ?-actin/ HSP70/ CD81 Flow cytometry Hardware adjustments Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 120 Particle yield Not reported EM EM-type Cryo-EM Image type Wide-field EV concentration Not

	EV210035	2/2	Homo sapiens	Cell culture supernatant	(d)(U)C DG Filtration	van Dommelen, Susan M	2016	78%
Study summary Full title Cetuximab treatment alters the content of extracellular vesicles released from tumor cells All authors Susan M van Dommelen, Roy van der Meel, Wouter W van Solinge, Maria Coimbra, Pieter Vader, Raymond M Schiffelers Journal Nanomedicine Abstract Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their co (show more...)Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). Materials & methods: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. Results: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. Conclusion: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior. Keywords: EGFR; biomarker; cancer therapy; cetuximab; diagnostics; exosomes; extracellular vesicles. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name A-431 Sample origin Cetuximab Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: TSG101/ Alix/ CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ Cetuximab non-EV: Lamin-A/ Lamin-C/ ATP5-A/ Tom20 Proteomics no EV density (g/ml) 1.10-1.21 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Cetuximab EV-producing cells A-431 EV-harvesting Medium Serum free medium Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type JA-30.50 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Density medium Sucrose Type Continuous Lowest density fraction 0.4 Highest density fraction 2.5 Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Top-down Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: duration (min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ Cetuximab/ TSG101/ Alix Not detected contaminants Lamin-A/ Lamin-C/ ATP5-A/ Tom20 Flow cytometry Hardware adjustments Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV200179	4/4	Homo sapiens	Blood plasma	(d)(U)C Filtration DG UF PEG precipitaton Gelatin-sepharose chromatograhy	Ouyang, Yingshi	2016	75%
Study summary Full title Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity All authors Yingshi Ouyang, Avraham Bayer, Tianjiao Chu, Vladimir A Tyurin, Valerian E Kagan, Adrian E Morelli, Carolyn B Coyne, Yoel Sadovsky Journal Placenta Abstract Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among (show more...)Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. (hide) EV-METRIC 75% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Density gradient Ultrafiltration PEG precipitaton Gelatin-sepharose chromatograhy Protein markers EV: CD63 non-EV: Fibronectin Proteomics yes EV density (g/ml) 1.08-1.10 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Healthy pregnant Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Density gradient Density medium Iodixanol Type Continuous Lowest density fraction 6 Highest density fraction 30 Total gradient volume, incl. sample (mL) 14 Sample volume (mL) 0.5 Orientation Top-down Rotor type Not specified Speed (g) 10000 Duration (min) Overnight Fraction volume (mL) . Fraction processing Ultrafiltration Pelleting: volume per fraction . Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 kda Membrane type PES Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63 Not detected contaminants Fibronectin Flow cytometry Hardware adjustments Proteomics Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mode Reported size (nm) 80-90nm EV concentration Yes

	EV210101	4/6	Homo sapiens	Urine	(d)(U)C Filtration SEC (non-commercial)	Welton, Joanne Louise	2016	67%
Study summary Full title Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array All authors Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton Journal J Extracell Vesicles Abstract Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide) EV-METRIC 67% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: Alix/ TSG101/ LAMP2A non-EV: Albumin Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Equal to or above 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 200000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 2.8 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Other;Spectrophotometry Western Blot Detected EV-associated proteins Alix/ LAMP2A/ TSG101 Not detected contaminants Albumin Flow cytometry Hardware adjustments Detected EV-associated proteins SOMAscan multiplex assay Characterization: Particle analysis NTA Report type Mean Reported size (nm) 117.75 EV concentration Yes Particle yield As the number of particles per µg protein;Yes, other: 20000000000 EM EM-type Cryo-EM Image type Close-up

	EV200134	2/2	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Salomon, Carlos	2016	67%
Study summary Full title Gestational Diabetes Mellitus Is Associated With Changes in the Concentration and Bioactivity of Placenta-Derived Exosomes in Maternal Circulation Across Gestation All authors Carlos Salomon, Katherin Scholz-Romero, Suchismita Sarker, Emma Sweeney, Miharu Kobayashi, Paula Correa, Sherri Longo, Gregory Duncombe, Murray D Mitchell, Gregory E Rice, Sebastian E Illanes Journal Diabetes Abstract Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) d (show more...)Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) during pregnancy, the exosomal profile in pregnancies complicated by gestational diabetes mellitus (GDM) remains to be established. The aim of this study was to compare the gestational-age profile of PdEs in maternal plasma of GDM with normal pregnancies and to determine the effect of exosomes on cytokine release from human umbilical vein endothelial cells. A prospective cohort of patients was sampled at three time points during pregnancy for each patient (i.e., 11-14, 22-24, and 32-36 weeks' gestation). A retrospective stratified study design was used to quantify exosomes present in maternal plasma of normal (n = 13) and GDM (n = 7) pregnancies. Gestational age and pregnancy status were identified as significant factors contributing to variation in plasma exosome concentration (ANOVA, P < 0.05). Post hoc analyses established that PdE concentration increased during gestation in both normal and GDM pregnancies; however, the increase was significantly greater in GDM (∼2.2-fold, ∼1.5-fold, and ∼1.8-fold greater at each gestational age compared with normal pregnancies). Exosomes isolated from GDM pregnancies significantly increased the release of proinflammatory cytokines from endothelial cells. Although the role of exosomes during GDM remains to be fully elucidated, exosome profiles may be of diagnostic utility for screening asymptomatic populations. (hide) EV-METRIC 67% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Gestational diabetes mellitus Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: PLAP/ CD63/ TSG101 non-EV: None Proteomics no EV density (g/ml) 1.122-1.156 Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Gestational diabetes mellitus Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 14.5mL Sample volume (mL) 0.5mL Orientation Top-down Rotor type T-8100 Speed (g) 100000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ TSG101 ELISA Detected EV-associated proteins PLAP Flow cytometry Hardware adjustments Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100 - 108nm EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV160004	2/5	Equus caballus	Synovial fluid	DG (d)(U)C	Boere J	2016	66%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 66% (60th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 71.08 (pelleting) Protein markers EV: Annexin-A1/ CD90/Thy1.1 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type MLS-50 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 71.08 Density gradient Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0M Highest density fraction 1.4M Sample volume (mL) 1.5 Orientation Bottom-up (sample migrates upwards) Rotor type MLS-50 Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 60 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 138.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Annexin-A1, CD90/Thy1.1 Characterization: Particle analysis EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 20-200 Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.

	EV160004	5/5	Equus caballus	Synovial fluid	DG (d)(U)C	Boere J	2016	66%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 66% (60th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 1421 (pelleting) Protein markers EV: Annexin-A1/ CD90/Thy1.1 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type MLS-50 Pelleting: speed (g) 10000 Pelleting: adjusted k-factor 1421. Density gradient Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0M Highest density fraction 1.4M Sample volume (mL) 1.5 Orientation Bottom-up (sample migrates upwards) Rotor type MLS-50 Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 60 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 138.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Annexin-A1, CD90/Thy1.1 Characterization: Particle analysis EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 20-200 Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.

	EV200044	1/1	Homo sapiens	Blood plasma	Precipitation DG	Zhang YN	2016	63%
Study summary Full title Extracellular Vesicle Proteins Associated with Systemic Vascular Events Correlate with Heart Failure: An Observational Study in a Dyspnoea Cohort. All authors Zhang YN, Vernooij F, Ibrahim I, Ooi S, Gijsberts CM, Schoneveld AH, Sen KW, den Ruijter HM, Timmers L, Richards AM, Jong CT, Mazlan I, Wang JW, Lam CS, de Kleijn DP Journal PLoS One Abstract BACKGROUND: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma (show more...)BACKGROUND: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma extracellular vesicle (EV) -levels of these proteins have been reported to be associated with systemic vascular events. Evidence is accumulating that inflammatory processes may play a pivotal role both in systemic vascular events and in heart failure. Therefore, we studied the association between plasma extracellular vesicle SerpinF2-, SerpinG1-, CystatinC and CD14-levels and the occurrence of acute heart failure in patients. METHODS AND RESULT: Extracellular vesicle protein levels of SerpinG1, SerpinF2, CystatinC and CD14 were measured in an observational study of 404 subjects presenting with dysponea at the emergency department (4B-cohort). Plasma extracellular vesicles were precipitated in a total extracellular vesicles (TEX)-fraction and in separate LDL- and HDL-subfractions. Extracellular vesicle protein levels were measured with a quantitative immune assay in all 3 precipitates. Out of 404 subjects, 141 (35%) were diagnosed with acutely decompensated heart failure. After correction for confounders (including comorbidities and medications), levels of CD14 in the HDL-fraction (OR 1.53, p = 0.01), SerpinF2 in the TEX-and LDL-fraction (ORs respectively 0.71 and 0.65, p<0.05) and SerpinG1 in the TEX-fraction (OR 1.55, p = 0.004) were statistically significantly related to heart failure. Furthermore, extracellular vesicle CD14- and SerpinF2-levels were significantly higher in heart failure patients with preserved ejection fraction than in those with reduced ejection fraction. CONCLUSION: Extracellular vesicle levels of CD14, SerpinG1 and SerpinF2 are associated with the occurrence of heart failure in subjects suspected for acute heart failure, suggesting common underlying pathophysiological mechanisms for heart failure and vascular events. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 test subject Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Precipitation Density gradient Protein markers EV: CD14/ SerpinC1/ SerpinG1/ CystC/ SerpinF2 non-EV: None Proteomics no EV density (g/ml) 1.02-1.17 Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Healthy test subject Separation Method Density gradient Only used for validation of main results Yes Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 8 Pelleting: duration (min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD14/ SerpinC1/ SerpinG1/ CystC/ SerpinF2 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-120

	EV210035	1/2	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	van Dommelen, Susan M	2016	56%
Study summary Full title Cetuximab treatment alters the content of extracellular vesicles released from tumor cells All authors Susan M van Dommelen, Roy van der Meel, Wouter W van Solinge, Maria Coimbra, Pieter Vader, Raymond M Schiffelers Journal Nanomedicine Abstract Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their co (show more...)Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). Materials & methods: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. Results: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. Conclusion: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior. Keywords: EGFR; biomarker; cancer therapy; cetuximab; diagnostics; exosomes; extracellular vesicles. (hide) EV-METRIC 56% (85th 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 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 Cell Name A-431 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Alix/ Cetuximab/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ CD9 non-EV: Lamin-A/ Lamin-C/ ATP5-A/ Tom20 Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells A-431 EV-harvesting Medium Serum free medium Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type JA-30.50 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ TSG101/ Alix Not detected EV-associated proteins Cetuximab Not detected contaminants Lamin-A/ Lamin-C/ ATP5-A/ Tom20 Flow cytometry Hardware adjustments Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV160008	1/3	Rattus norvegicus	Cell culture supernatant	(d)(U)C	Cianciaruso C	2016	55%
Study summary Full title Primary Human and Rat β-Cells Release the Intracellular Autoantigens GAD65, IA-2, and Proinsulin in Exosomes Together With Cytokine-Induced Enhancers of Immunity. All authors Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S Journal Diabetes Abstract The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide) EV-METRIC 55% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name Primary pancreatic islets 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: GAD67/ GAD65/ Insulin/ PDI/ Flotillin-1/ CD9/ IA-2 non-EV: Gp96/ Calreticulin Proteomics yes Show all info Study aim Function, Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Primary pancreatic islets EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 87.5 Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, Flotillin-1, GAD65, GAD67, IA-2, PDI Not detected contaminants Calreticulin, Gp96 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Proteomics Proteomics database No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 139 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 120

	EV160005	1/2	Homo sapiens	Bronchoalveolar lavage fluid	(d)(U)C Filtration	Héliot A	2016	55%
Study summary Full title Smoker extracellular vesicles influence status of human bronchial epithelial cells. All authors Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ Journal Int J Hyg Environ Health Abstract Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide) EV-METRIC 55% (33rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Bronchoalveolar lavage fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 126 (pelleting) / 126 (washing) Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 126.0 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 126.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Characterization: Particle analysis NTA Report type Size range/distribution;Mean Reported size (nm) 138.1±2.2 EV concentration Yes Particle yield 2.48E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Extra information UC rotors added to the EV-TRACK entry after publication

	EV160005	2/2	Homo sapiens	Bronchoalveolar lavage fluid	(d)(U)C Filtration	Héliot A	2016	55%
Study summary Full title Smoker extracellular vesicles influence status of human bronchial epithelial cells. All authors Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ Journal Int J Hyg Environ Health Abstract Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide) EV-METRIC 55% (33rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Bronchoalveolar lavage fluid Sample origin Smokers Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 126 (pelleting) / 126 (washing) Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Sample Condition Smokers Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 126.0 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 126.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Characterization: Particle analysis NTA Report type Size range/distribution;Mean Reported size (nm) 139.8±3.3 EV concentration Yes Particle yield 1.94E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Extra information UC rotors added to the EV-TRACK entry after publication

	EV160004	1/5	Equus caballus	Synovial fluid	DG (d)(U)C	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (35th 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 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 Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 83.68 (pelleting) Protein markers EV: CD44/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation Method Differential ultracentrifugation 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 Equal to or above 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 65 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 83.68 Density gradient Density medium Iodixanol Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV160004	3/5	Equus caballus	Synovial fluid	DG (d)(U)C	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (35th 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 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 Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 167.3 (pelleting) Protein markers EV: CD44/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 65 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 167.3 Density gradient Density medium Iodixanol Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV160004	4/5	Equus caballus	Synovial fluid	DG (d)(U)C	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (35th 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 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 Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 1673 (pelleting) Protein markers EV: CD44/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Obtain an EV pellet : Yes Pelleting: time(min) 35 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 10000 Pelleting: adjusted k-factor 1673. Density gradient Density medium Iodixanol Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV210101	3/6	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Welton, Joanne Louise	2016	50%
Study summary Full title Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array All authors Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton Journal J Extracell Vesicles Abstract Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide) EV-METRIC 50% (85th 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 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 exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD81/ CD9 non-EV: Albumin/ ApoB Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Equal to or above 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-110 Pelleting: speed (g) 200000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Other;Spectrophotometry ELISA Detected EV-associated proteins CD81/ CD9 Detected contaminants ApoB Flow cytometry Hardware adjustments Detected EV-associated proteins SOMAscan multiplex assay Characterization: Particle analysis NTA Report type Mean Reported size (nm) 86.47 EV concentration Yes Particle yield As the number of particles per µg protein;Yes, other: 5000000000 EM EM-type Cryo-EM Image type Close-up

	EV160007	1/3	Homo sapiens	Blood plasma	(d)(U)C Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide) EV-METRIC 50% (85th 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 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 Acute myeloid leukemia Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration SEC Protein markers EV: TSG101/ PD-1/ CD123/ CD96/ CD44/ CD34/ Pro-TGFbeta1 LAP/ Pro-TGFbeta1LAP/ PD-L1/ CLL-1/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Acute myeloid leukemia Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 66 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101, CD44, CD34, CD123, CD96, CLL-1, Pro-TGFbeta1 LAP, PD-1, PD-L1 Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 77-92 EV concentration Yes Particle yield 8.90E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV160007	2/3	Homo sapiens	Blood plasma	(d)(U)C Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide) EV-METRIC 50% (85th 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 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 Head and neck squamous cell carcinoma Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration SEC Protein markers EV: TSG101/ CD39/ PD-1/ CD73/ Cox2/ Fas/ FasL/ PD-L1/ HSP70/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Head and neck squamous cell carcinoma Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 123 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-1, PD-L1 Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 73-76 EV concentration Yes Particle yield 1.10E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV160007	3/3	Homo sapiens	Blood plasma	(d)(U)C Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide) EV-METRIC 50% (85th 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 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 exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration SEC Protein markers EV: TSG101/ CD39/ CD123/ PD-1/ CD73/ Cox2/ Fas/ Pro-TGFbeta1 LAP/ Pro-TGFbeta1LAP/ FasL/ PD-L1/ CLL-1/ HSP70/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 32 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101, CD123, CLL-1, Pro-TGFbeta1 LAP, PD-1, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-L1 Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 69-76 EV concentration Yes Particle yield 7.00E+09 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV160000	1/1	Homo sapiens	Milk	DG (d)(U)C	van Herwijnen MJ	2016	50%
Study summary Full title Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components. All authors van Herwijnen MJ, Zonneveld MI, Goerdayal S, Nolte-'t Hoen EN, Garssen J, Stahl B, Maarten Altelaar AF, Redegeld FA, Wauben MH Journal Mol Cell Proteomics Abstract Breast milk contains several macromolecular components with distinctive functions, whereby milk fat (show more...)Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. (hide) EV-METRIC 50% (63rd 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Flotillin-1/ CD9 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Milk Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Density gradient Density medium Sucrose Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 6.5 Orientation Top-down (sample migrates downwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 38.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis PMID previous EV protein analysis 25206958 Extra characterization Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, Flotillin-1 Proteomics Proteomics database Yes PMID previous EV particle analysis 25206958 Extra information A different gradient protocol was used in the publication of the same group that was referred to for additional protein and particle analysis of milk-derived extracellular vesicles (PMID: 25206958).

	EV210034	1/14	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	Au Yeung, Chi Lam	2016	45%
Study summary Full title Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1 All authors Chi Lam Au Yeung, Ngai-Na Co, Tetsushi Tsuruga, Tsz-Lun Yeung, Suet-Ying Kwan, Cecilia S Leung, Yong Li, Edward S Lu, Kenny Kwan, Kwong-Kwok Wong, Rosemarie Schmandt, Karen H Lu, Samuel C Mok Journal Nat Commun Abstract Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the (show more...)Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer. (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 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 Cell Name Primary cancer-associated adipocytes 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: HSP70/ CD63 non-EV: GM130 Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Primary cancer-associated adipocytes EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 90 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ HSP70 Not detected contaminants GM130 Flow cytometry Hardware adjustments Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 70-130 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210139	1/6	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name U87 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells U87 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µmNo Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Not detected contaminants Calnexin Flow cytometry Hardware adjustments Proteomics Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	2/6	Homo sapiens	Cell culture supernatant	(d)(U)C	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name U87 Sample origin Control condition Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells U87 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Obtain an EV pellet : Yes Pelleting: time(min) 30 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Detected contaminants Calnexin Flow cytometry Hardware adjustments Proteomics Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-600 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	3/6	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name Huh7 Sample origin Control condition Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Huh7 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µmNo Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ Tsg101 Not detected contaminants Calnexin Flow cytometry Hardware adjustments Proteomics Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	4/6	Homo sapiens	Cell culture supernatant	(d)(U)C	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name Huh7 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Huh7 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Obtain an EV pellet : Yes Pelleting: time(min) 30 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Detected contaminants Calnexin Flow cytometry Hardware adjustments Proteomics Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-750 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	5/6	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name Bone marrow-derived mesenchymal stem cells Sample origin Control condition Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Bone marrow-derived mesenchymal stem cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µmNo Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Not detected contaminants Calnexin Flow cytometry Hardware adjustments Proteomics Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes Particle yield Not reported NA EM EM-type Transmission-EM Image type Wide-field

	EV210139	6/6	Homo sapiens	Cell culture supernatant	(d)(U)C	Haraszti, Reka A	2016	44%
Study summary Full title High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources All authors Reka A Haraszti, Marie-Cecile Didiot, Ellen Sapp, John Leszyk, Scott A Shaffer, Hannah E Rockwell, Fei Gao, Niven R Narain, Marian DiFiglia, Michael A Kiebish, Neil Aronin, Anastasia Khvorova Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in di (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name Bone marrow-derived mesenchymal stem cells 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Bone marrow-derived mesenchymal stem cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Obtain an EV pellet : Yes Pelleting: time(min) 30 Pelleting: rotor type Not specified Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Tsg101 Detected contaminants Calnexin Flow cytometry Hardware adjustments Proteomics Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-750 EV concentration Yes Particle yield Not reported EM EM-type Transmission-EM Image type Wide-field

	EV210099	1/9	Homo sapiens	Cell culture supernatant	(d)(U)C	Rider, Mark A	2016	44%
Study summary Full title ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles All authors Mark A Rider, Stephanie N Hurwitz, David G Meckes Jr Journal Sci Rep Abstract Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known (show more...)Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name HEK293 T 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ TSG101/ HSP70/ Alix non-EV: None Proteomics no Show all info Study aim New methodological development/ Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells HEK293 T EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 70 Wash: Rotor Type TLA120.2 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD63/ TSG101/ HSP70/ Alix Flow cytometry Hardware adjustments Characterization: Particle analysis NTA Report type Not Reported Particle yield NA NA EM EM-type Transmission-EM Image type Close-up Report size (nm) Not specified

	EV200178	2/4	Homo sapiens	Blood plasma	(d)(U)C DC Filtration	Pillay, Preenan	2016	44%
Study summary Full title Placental exosomes and pre-eclampsia: Maternal circulating levels in normal pregnancies and, early and late onset pre-eclamptic pregnancies All authors Preenan Pillay, Niren Maharaj, Jagidesa Moodley, Irene Mackraj Journal Placenta Abstract Introduction and aim: Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biolog (show more...)Introduction and aim: Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biological cells under normal and pathological conditions. Although there have been reports of circulating exosomes in normal pregnancy, the relevance of placental-derived exosomes in normal and abnormal pregnancies still needs to be elucidated. The aim of this study was to quantify total and placental-derived exosomes in maternal plasma from normal (N), early onset- and late onset-preeclampsia (PE). Method: Plasma samples were obtained from pregnant women in the third trimester, for the isolation of exosomes by differential ultracentrifugation. Total exosomes were quantified using nanoparticle tracking analysis and immuno-reactive exosomal CD63 quantification. Placental-derived exosomes were quantified using placental alkaline phosphatase (PLAP) as a specific marker. The contribution of placental-derived exosomes to total exosomes in maternal plasma was determined by the ratio of PLAP+ exosomes to CD63+ exosomes. Results: The concentration of total exosomes significantly increased in early onset-PE and late onset-PE compared to N (≤33 weeks) and N (≥34 weeks). The relative concentration of placental-derived exosomes significantly increased in early onset-PE but decreased in late onset-PE compared to N. The ratio of PLAP+ exosomes to total number of exosomes significantly decreased in early onset-PE and late onset-PE. A positive correlation between total and placental-derived exosomes were obtained in N (≤33 weeks: Pearson's r = 0.60, ≥34 weeks: Pearson's r = 0.67) and early onset-PE (Pearson's r = 0.51, p < 0.05) with the inverse in late onset-PE (Pearson's r = -0.62, p < 0.01). Conclusion: The differences in the contribution of placental-derived exosomes to total exosomes in maternal circulation suggests a possible pathophysiological role of placental-derived exosomes in pre-eclampsia. (hide) EV-METRIC 44% (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 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 (>34 weeks gestation) Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Filtration Protein markers EV: PLAP/ CD63 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Normal pregnancy (>34 weeks gestation) Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type MLA-55 Pelleting: speed (g) 110000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type MLA-55 Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method Lowry Western Blot Detected EV-associated proteins CD63 ELISA Detected EV-associated proteins CD63/ PLAP Flow cytometry Hardware adjustments Characterization: Particle analysis NTA Report type Mean Reported size (nm) 100.3 + - 7.78 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV160009	1/2	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	Benedikter BJ	2016	44%
Study summary Full title Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants. All authors Benedikter BJ, Volgers C, van Eijck PH, Wouters EFM, Savelkoul PHM, Reynaert NL, Haenen GRMM, Rohde GGU, Weseler AR, Stassen FRM Journal J Cell Sci Abstract INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) wi (show more...)INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name BEAS2B Sample origin Exposed to cigarette smoke extract Focus vesicles extracellular vesicle / exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 133.2 (pelleting) Protein markers EV: CD63 non-EV: grp94 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Exposed to cigarette smoke extract EV-producing cells BEAS2B EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability 75 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 150 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 117734 Pelleting: adjusted k-factor 133.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected contaminants grp94 Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 60-300 EV concentration Yes EM EM-type Cryo-EM Image type Close-up Report size (nm) 50-160

	EV160009	2/2	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	Benedikter BJ	2016	44%
Study summary Full title Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants. All authors Benedikter BJ, Volgers C, van Eijck PH, Wouters EFM, Savelkoul PHM, Reynaert NL, Haenen GRMM, Rohde GGU, Weseler AR, Stassen FRM Journal J Cell Sci Abstract INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) wi (show more...)INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name BEAS2B Sample origin Control condition Focus vesicles extracellular vesicle / exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 133.2 (pelleting) Protein markers EV: CD63 non-EV: grp94 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells BEAS2B EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability 100 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 150 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 117734 Pelleting: adjusted k-factor 133.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected contaminants grp94 Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 60-300 EV concentration Yes EM EM-type Cryo-EM Image type Close-up Report size (nm) 50-160

	EV160008	2/3	Homo sapiens	Cell culture supernatant	(d)(U)C	Cianciaruso C	2016	44%
Study summary Full title Primary Human and Rat β-Cells Release the Intracellular Autoantigens GAD65, IA-2, and Proinsulin in Exosomes Together With Cytokine-Induced Enhancers of Immunity. All authors Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S Journal Diabetes Abstract The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide) EV-METRIC 44% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods 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 Cell Name Primary human pancreatic islets, primary rat pancreatic islets, INS1 cell line 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Insulin/ GAD65/ Flotillin-1/ CD9/ IA-2 non-EV: None Proteomics yes Show all info Study aim Function, Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Primary human pancreatic islets, primary rat pancreatic islets, INS1 cell line EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 87.5 Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, Flotillin-1, GAD65, IA-2 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Proteomics Proteomics database No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 143 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 120

	EV210099	2/9	Homo sapiens	Cell culture supernatant	DC	Rider, Mark A	2016	38%
Study summary Full title ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles All authors Mark A Rider, Stephanie N Hurwitz, David G Meckes Jr Journal Sci Rep Abstract Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known (show more...)Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes. (hide) EV-METRIC 38% (68th 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 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 Cell Name HEK293 T 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DC Protein markers EV: CD63/ TSG101/ HSP70/ Alix non-EV: None Proteomics no Show all info Study aim New methodological development/ Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells HEK293 T EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Density cushion Density medium Sucrose Sample volume 4 Cushion volume 35 Density of the cushion 30% Centrifugation time 75 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD63/ TSG101/ HSP70/ Alix Flow cytometry Hardware adjustments Characterization: Particle analysis NTA Report type Not Reported Particle yield NA NA EM EM-type Transmission-EM Image type Close-up Report size (nm) Not specified

	EV210099	5/9	Homo sapiens	Cell culture supernatant	NA	Rider, Mark A	2016	38%
Study summary Full title ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles All authors Mark A Rider, Stephanie N Hurwitz, David G Meckes Jr Journal Sci Rep Abstract Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known (show more...)Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes. (hide) EV-METRIC 38% (68th 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 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 Cell Name HEK293 T 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture NA Protein markers EV: CD63/ TSG101/ HSP70/ Alix non-EV: None Proteomics yes Show all info Study aim New methodological development/ Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells HEK293 T EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Other Name other separation method ExtraPEG Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Alix/ TSG101/ HSC70/ CD63 Flow cytometry Hardware adjustments Proteomics Proteomics database

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