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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
	EV200137	3/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (52nd 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 Pre-eclampsia 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 Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Pre-eclampsia Separation Method Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Flow cytometry Hardware adjustments

	EV200137	4/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated annexin AV-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (52nd 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 Pre-eclampsia 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 Streptavidin bead precipitation of biotinylated annexin AV-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Pre-eclampsia Separation Method Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Flow cytometry Hardware adjustments

	EV170005	3/4	Homo sapiens	Cell culture supernatant	(d)(U)C	Suárez H	2017	25%
Study summary Full title A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles. All authors Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M Journal Sci Rep Abstract Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide) EV-METRIC 25% (51st 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 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 (d)(U)C Adj. k-factor 264.9 (pelleting) / 264.9 (washing) Protein markers EV: CD59/ CD9 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells SK-MEL103 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) 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) 120 Pelleting: rotor type AH-627 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 264.9 Wash: time (min) 120 Wash: Rotor Type AH-627 Wash: speed (g) 100000 Wash: adjusted k-factor 264.9 Characterization: Protein analysis Protein Concentration Method BCA Characterization: Particle analysis NTA Report type Median

	EV170042	1/4	Homo sapiens	Urine	(d)(U)C	Silvers CR	2017	22%
Study summary Full title Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. All authors Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF Journal J Cell Sci Abstract The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are need (show more...)The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation. (hide) EV-METRIC 22% (45th 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 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 (d)(U)C Protein markers EV: Alix/ Annexin VII/ EHD4/ AnnexinVII non-EV: None Proteomics no Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: speed (g) 20000 Wash: time (min) 70 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Detected EV-associated proteins Alix, Annexin VII, EHD4

	EV170042	2/4	Homo sapiens	Cell culture supernatant	(d)(U)C	Silvers CR	2017	22%
Study summary Full title Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. All authors Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF Journal J Cell Sci Abstract The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are need (show more...)The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation. (hide) EV-METRIC 22% (45th 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 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 (d)(U)C Protein markers EV: Alix/ TSG101/ CD9 non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells TCCSUP EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: speed (g) 20000 Wash: time (min) 70 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Detected EV-associated proteins Alix, CD9, TSG101 Proteomics Proteomics database No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 35-300

	EV170042	3/4	Homo sapiens	Urine	(d)(U)C	Silvers CR	2017	22%
Study summary Full title Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. All authors Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF Journal J Cell Sci Abstract The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are need (show more...)The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation. (hide) EV-METRIC 22% (45th 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 Bladder cancer 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 (d)(U)C Protein markers EV: SNB1/ EHD4/ Annexin VII/ HEXB/ Alix/ S100A4/ AnnexinVII non-EV: None Proteomics no Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Sample Condition Bladder cancer Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: speed (g) 20000 Wash: time (min) 70 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Detected EV-associated proteins Alix, Annexin VII, EHD4, HEXB, S100A4, SNB1 Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV170072	1/5	Homo sapiens	Cell culture supernatant	(d)(U)C	Nabet BY	2017	14%
Study summary Full title Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer. All authors Nabet BY, Qiu Y, Shabason JE, Wu TJ, Yoon T, Kim BC, Benci JL, DeMichele AM, Tchou J, Marcotrigiano J, Minn AJ. Journal Cell Abstract Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, t (show more...)Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT. (hide) EV-METRIC 14% (36th 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 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 (d)(U)C Protein markers EV: None non-EV: None 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 MRC5 EV-harvesting Medium EV-depleted medium Preparation of EDS 3h at 100,000g;Other preparation 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 Not specified Pelleting: speed (g) 100000 Protein Concentration Method Lowry-based assay Characterization: Particle analysis

	EV170072	2/5	Homo sapiens	Cell culture supernatant	(d)(U)C	Nabet BY	2017	14%
Study summary Full title Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer. All authors Nabet BY, Qiu Y, Shabason JE, Wu TJ, Yoon T, Kim BC, Benci JL, DeMichele AM, Tchou J, Marcotrigiano J, Minn AJ. Journal Cell Abstract Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, t (show more...)Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT. (hide) EV-METRIC 14% (36th 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 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 (d)(U)C Protein markers EV: None non-EV: None 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 MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS 3h at 100,000g;Other preparation 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 Not specified Pelleting: speed (g) 100000 Protein Concentration Method Lowry-based assay Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133.4 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV180054	1/2	Homo sapiens	Urine	(d)(U)C SEC UF	Lozano-Ramos SI	2017	14%
Study summary Full title Molecular profile of urine extracellular vesicles from normo-functional kidneys reveal minimal differences between living and deceased donors. All authors Lozano-Ramos SI, Bancu I, Carreras-Planella L, Monguió-Tortajada M, Cañas L, Juega J, Bonet J, Armengol MP, Lauzurica R, Borràs FE Journal BMC Nephrol Abstract BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney disease (show more...)BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney diseases leading to irreversible kidney failure. Considering the origin of the graft, several studies have reported differences between living (LD) and deceased donors (DD) in graft and patient survival. These differences seem to be related to multiple factors including, donor age and time of cold ischemia among others. Many of transplanted organs come from old-aged DDs, in which pre-transplant biopsy is recommended. However, kidney biopsy has several limitations, and there is a need to develop alternatives to assess the status of a kidney before transplantation. As the analysis of urinary extracellular vesicles (uEVs) rendered promising results as non-invasive biomarkers of kidney-related pathologies, this pilot study aimed to investigate whether profiling uEVs of LDs and DDs may be of help to assess the quality of the kidney before nephrectomy. METHODS: uEVs from 5 living donors and 7 deceased donors were isolated by size-exclusion chromatography, and their protein and miRNA content were analysed by liquid chromatography followed by mass spectrometry and next generation sequencing, respectively. Then, hierarchical clustering and venn diagrams were done with Perseus software and InteractiVenn tool. Specific EVs data bases were also used for Gene Ontology analysis. RESULTS: Next generation sequencing revealed that uEVs from DDs contained less miRNAs than LDs, but most of the DD-expressed miRNAs were shared with LDs (96%). Only miR-326 (targeting the apoptotic-related Bcl2) was found significantly over-represented in LD. Focusing on the protein content, we detected a low intra-group correlation in both types of donors. Despite these differences, hierarchical clustering of either miRNA or protein data could not identify a differential profile between LDs and DDs. Of note, 90% of transplanted patients had a functional graft after a year from KTx. CONCLUSIONS: In this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs. (hide) EV-METRIC 14% (36th 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 Deceased 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 (d)(U)C + SEC + UF Protein markers EV: CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Sample Condition Deceased Separation Method Differential ultracentrifugation centrifugation steps Between 10,000 g and 50,000 g Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 0.3 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Nanodrop Proteomics Proteomics database No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 124-250 EV concentration Yes

	EV180054	2/2	Homo sapiens	Urine	(d)(U)C SEC UF	Lozano-Ramos SI	2017	14%
Study summary Full title Molecular profile of urine extracellular vesicles from normo-functional kidneys reveal minimal differences between living and deceased donors. All authors Lozano-Ramos SI, Bancu I, Carreras-Planella L, Monguió-Tortajada M, Cañas L, Juega J, Bonet J, Armengol MP, Lauzurica R, Borràs FE Journal BMC Nephrol Abstract BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney disease (show more...)BACKGROUND: Kidney transplantation (KTx) is the best therapeutic approach for chronic kidney diseases leading to irreversible kidney failure. Considering the origin of the graft, several studies have reported differences between living (LD) and deceased donors (DD) in graft and patient survival. These differences seem to be related to multiple factors including, donor age and time of cold ischemia among others. Many of transplanted organs come from old-aged DDs, in which pre-transplant biopsy is recommended. However, kidney biopsy has several limitations, and there is a need to develop alternatives to assess the status of a kidney before transplantation. As the analysis of urinary extracellular vesicles (uEVs) rendered promising results as non-invasive biomarkers of kidney-related pathologies, this pilot study aimed to investigate whether profiling uEVs of LDs and DDs may be of help to assess the quality of the kidney before nephrectomy. METHODS: uEVs from 5 living donors and 7 deceased donors were isolated by size-exclusion chromatography, and their protein and miRNA content were analysed by liquid chromatography followed by mass spectrometry and next generation sequencing, respectively. Then, hierarchical clustering and venn diagrams were done with Perseus software and InteractiVenn tool. Specific EVs data bases were also used for Gene Ontology analysis. RESULTS: Next generation sequencing revealed that uEVs from DDs contained less miRNAs than LDs, but most of the DD-expressed miRNAs were shared with LDs (96%). Only miR-326 (targeting the apoptotic-related Bcl2) was found significantly over-represented in LD. Focusing on the protein content, we detected a low intra-group correlation in both types of donors. Despite these differences, hierarchical clustering of either miRNA or protein data could not identify a differential profile between LDs and DDs. Of note, 90% of transplanted patients had a functional graft after a year from KTx. CONCLUSIONS: In this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs. (hide) EV-METRIC 14% (36th 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 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 (d)(U)C + SEC + UF Protein markers EV: CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 10,000 g and 50,000 g Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 0.3 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Nanodrop Proteomics Proteomics database No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 124-250 EV concentration Yes

	EV170042	4/4	Homo sapiens	Cell culture supernatant	(d)(U)C	Silvers CR	2017	14%
Study summary Full title Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. All authors Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF Journal J Cell Sci Abstract The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are need (show more...)The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation. (hide) EV-METRIC 14% (36th 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 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells SVHUC EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: speed (g) 20000 Wash: time (min) 70 Wash: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics Proteomics database No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 35-300

	EV170036	1/12	Homo sapiens	Blood plasma	(d)(U)C	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (36th 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 Prostate cancer Focus vesicles EV12 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Prostate cancer 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) 90 Pelleting: speed (g) 12000 Protein Concentration Method microBCA Protein Concentration 311 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 1.55E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	2/12	Homo sapiens	Serum	(d)(U)C	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (59th 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 Serum Sample origin Control condition Focus vesicles EV12 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum 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) 90 Pelleting: speed (g) 12000 Protein Concentration Method microBCA Protein Concentration 11.4 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 1.34E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	3/12	Homo sapiens	Blood plasma	(d)(U)C Filtration	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (36th 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 Prostate cancer Focus vesicles EV120 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 (d)(U)C + Filtration Adj. k-factor 213.2 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Prostate cancer Separation Method Differential ultracentrifugation centrifugation steps 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 SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes we proceeded with IR and RAMAN analysis of EVs isolated by 12000 x g (frequently designated as micro Protein Concentration Method microBCA Protein Concentration 4-6 for healty donors in EV12; 100 in cancer patients; Characterization: Particle analysis Other particle analysis name(1) Raman spectroscopy

	EV170036	4/12	Homo sapiens	Blood plasma	(d)(U)C	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (36th 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 Prostate cancer Focus vesicles EV120 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Prostate cancer 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) 90 Pelleting: speed (g) 120000 Protein Concentration Method microBCA Protein Concentration 311 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 4.28E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	5/12	Homo sapiens	Blood plasma	(d)(U)C	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (36th 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 EV120 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker 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 Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: speed (g) 120000 Protein Concentration Method microBCA Protein Concentration 9.4 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 1.86E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	6/12	Homo sapiens	Serum	(d)(U)C	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (59th 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 Serum Sample origin Control condition Focus vesicles EV120 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum 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) 90 Pelleting: speed (g) 120000 Protein Concentration Method microBCA Protein Concentration 1273 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 1.69E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	7/12	Homo sapiens	Serum	(d)(U)C	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (59th 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 Serum Sample origin Prostate cancer Focus vesicles EV120 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Prostate cancer 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) 90 Pelleting: speed (g) 120000 Protein Concentration Method microBCA Protein Concentration 839.6 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 4.84E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	9/12	Homo sapiens	Blood plasma	(d)(U)C Filtration	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (36th 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 Benign prostate hyperplasia Focus vesicles EV12 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 (d)(U)C + Filtration Adj. k-factor 213.2 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Benign prostate hyperplasia Separation Method Differential ultracentrifugation centrifugation steps 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 SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes we proceeded with IR and RAMAN analysis of EVs isolated by 12000 x g (frequently designated as micro Protein Concentration Method microBCA Protein Concentration 4-6 for healty donors in EV12; 100 in cancer patients; Characterization: Particle analysis Other particle analysis name(1) Raman spectroscopy

	EV170036	11/12	Homo sapiens	Serum	(d)(U)C	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (59th 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 Serum Sample origin Prostate cancer Focus vesicles EV12 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Prostate cancer 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) 90 Pelleting: speed (g) 12000 Protein Concentration Method microBCA Protein Concentration 255.2 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 2.24E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	12/12	Homo sapiens	Blood plasma	(d)(U)C	Krafft C	2017	14%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 14% (36th 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 EV12 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker 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 Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: speed (g) 12000 Protein Concentration Method microBCA Protein Concentration 9.4 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EV concentration Yes Particle yield 4.26E+09 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170022	1/5	Streptococcus pneumoniae	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens. All authors Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal FEMS Microbiol Lett Abstract Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: Streptococcus pneumoniae antigen non-EV: None Proteomics no Show all info Study aim Function, Biogenesis/cargo sorting Sample Species Streptococcus pneumoniae Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Streptococcus pneumoniae EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) Streptococcus pneumoniae antigen Characterization: Particle analysis TRPS EV concentration Yes

	EV170022	2/5	Moraxella catarrhalis	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens. All authors Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal FEMS Microbiol Lett Abstract Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: Moraxella catarrhalis antigen non-EV: None Proteomics no Show all info Study aim Function, Biogenesis/cargo sorting Sample Species Moraxella catarrhalis Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Moraxella catarrhalis EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) Moraxella catarrhalis antigen Characterization: Particle analysis TRPS EV concentration Yes

	EV170022	3/5	Haemophilus influenzae	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens. All authors Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal FEMS Microbiol Lett Abstract Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: non-typeable Haemophilus influenzae antigen non-EV: None Proteomics no Show all info Study aim Function, Biogenesis/cargo sorting Sample Species Haemophilus influenzae Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells non-typeable Haemophilus influenzae EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) non-typeable Haemophilus influenzae antigen Characterization: Particle analysis TRPS EV concentration Yes

	EV170022	4/5	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens. All authors Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal FEMS Microbiol Lett Abstract Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: CD81/ CD63 non-EV: None Proteomics no Show all info Study aim Function, Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells THP1 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) CD63 Characterization: Particle analysis TRPS EV concentration Yes

	EV170022	5/5	Pseudomonas aeruginosa	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens. All authors Volgers C, Benedikter BJ, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal FEMS Microbiol Lett Abstract Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease (show more...)Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: Pseudomonas aeruginosa antigen non-EV: None Proteomics no Show all info Study aim Function, Biogenesis/cargo sorting Sample Species Pseudomonas aeruginosa Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Pseudomonas aeruginosa EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) Pseudomonas aeruginosa antigen Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	1/5	Haemophilus influenzae	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: non-typeable Haemophilus influenzae antigen non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Haemophilus influenzae Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells non-typeable Haemophilus influenzae EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) non-typeable Haemophilus influenzae antigen Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	2/5	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: CD81/ CD63 non-EV: None 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 THP1 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) CD63 Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	3/5	Streptococcus pneumoniae	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: Streptococcus pneumoniae antigen non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Streptococcus pneumoniae Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Streptococcus pneumoniae EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) Streptococcus pneumoniae antigen Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	4/5	Pseudomonas aeruginosa	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: Pseudomonas aeruginosa antigen non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Pseudomonas aeruginosa Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Pseudomonas aeruginosa EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) Pseudomonas aeruginosa antigen Characterization: Particle analysis TRPS EV concentration Yes

	EV170023	5/5	Moraxella catarrhalis	Cell culture supernatant	(d)(U)C Filtration SEC UF	Volgers C	2017	14%
Study summary Full title Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. All authors Volgers C, Grauls GE, Hellebrand PHM, Savelkoul PHM, Stassen FRM Journal Scientific Reports Abstract Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations (show more...)Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected. (hide) EV-METRIC 14% (36th 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 Sample origin Control condition Focus vesicles membrane 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 (d)(U)C + Filtration + SEC + UF Protein markers EV: Moraxella catarrhalis antigen non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Moraxella catarrhalis Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Moraxella catarrhalis EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Selected surface protein(s) Moraxella catarrhalis antigen Characterization: Particle analysis TRPS EV concentration Yes

	EV170005	2/4	Homo sapiens	Urine	(d)(U)C SEC UF	Suárez H	2017	14%
Study summary Full title A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles. All authors Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M Journal Sci Rep Abstract Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide) EV-METRIC 14% (36th 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 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 (d)(U)C + SEC + UF Protein markers EV: CD63/ CD9 non-EV: None Proteomics no Show all info Study aim New methodological development 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 Ultra filtration Cut-off size (kDa) Not spec Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA

	EV170066	5/5	Homo sapiens	Serum	ExoQuick	Zhang R	2017	13%
Study summary Full title Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in non-small cell lung cancer. All authors Zhang R, Xia Y, Wang Z, Zheng J, Chen Y, Li X, Wang Y, Ming H. Journal Biochem Biophys Res Commun Abstract Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), M (show more...)Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), MALAT-1 was first identified lncRNA that was related to lung cancer metastasis. However, the relationship between exosomal lncRNAs and the diagnosis and prognosis of NSCLC was poorly understood. The aim of this study is to evaluate the clinical significance of serum exosomal MALAT-1 as a biomarker in the metastasis of NSCLC. In this study, we firstly isolated the exosomes from healthy subjects and NSCLC patients. Then we measured the expression levels of MALAT-1 contained in exosomes, and found that exosomal MALAT-1 was highly expressed in NSCLC patients, more importantly, the levels of exosomal MALAT-1 were positively associated with tumor stage and lymphatic metastasis. In addition, we decreased MALAT-1 expression by short hairpin RNA and conducted a series of assays including MTT, cell cycle, colony formation, wound-healing scratch and Annexin/V PI by flow cytometry in human lung cancer cell lines. These in vitro studies demonstrated that serum exosome-derived long noncoding RNA MALAT-1 promoted the tumor growth and migration, and prevented tumor cells from apoptosis in lung cancer cell lines. Taken together, this study shed a light on utilizing MALAT-1 in exosomes as a non-invasive serum-based tumor biomarker for diagnosis and prognosis of NSCLC. (hide) EV-METRIC 13% (51st 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 Serum Sample origin non-small cell lung cancer 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 ExoQuick Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition non-small cell lung cancer Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV170072	5/5	Homo sapiens	Cell culture supernatant	PEG precipitation	Nabet BY	2017	13%
Study summary Full title Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer. All authors Nabet BY, Qiu Y, Shabason JE, Wu TJ, Yoon T, Kim BC, Benci JL, DeMichele AM, Tchou J, Marcotrigiano J, Minn AJ. Journal Cell Abstract Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, t (show more...)Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT. (hide) EV-METRIC 13% (31st 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 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 PEG precipitation Protein markers EV: None non-EV: None 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 MRC5 EV-harvesting Medium EV-depleted medium Preparation of EDS 3h at 100,000g;Other preparation Separation Method Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins TSG101/ beta-actin/ SRP9 Characterization: Particle analysis

	EV180048	1/1	Mus musculus	Cell culture supernatant	(d)(U)C	Luther KM	2017	11%
Study summary Full title Exosomal miR-21a-5p mediates cardioprotection by mesenchymal stem cells. All authors Luther KM, Haar L, McGuinness M, Wang Y, Lynch Iv TL, Phan A, Song Y, Shen Z, Gardner G, Kuffel G, Ren X, Zilliox MJ, Jones WK Journal J Mol Cell Cardiol Abstract Though experimental, stem cell transplantation has the potential to improve the condition of the hea (show more...)Though experimental, stem cell transplantation has the potential to improve the condition of the heart after myocardial infarction. It does so by reducing infarct size and inducing repair of heart muscle and its blood supply. Mesenchymal stem cells (MSC) have been found to be effective in pre-clinical animal models and clinical trials, but the mechanisms by which they induce cardioprotection and repair are still not fully understood. Small extracellular vesicles known as exosomes are now recognized to be key mediators of beneficial MSC paracrine effects, and the concept that they transfer miRNA to change gene expression in recipient cells is of current therapeutic interest. We present complete deep miRNA sequencing of MSC exosome cargo, and found that of several cardioprotective miRNAs, miR-21a-5p was the most abundant. Because miR-21a-5p is a well-known cardioprotective miRNA, we investigated the hypothesis that MSC exosomes can cardioprotect the heart by increasing the level of miR-21a-5p in recipient cardiac cells, thereby downregulating expression of the pro-apoptotic gene products PDCD4, PTEN, Peli1 and FasL in the myocardium. Using miR-21 mimic transfection and treatment with wild type and miR-21a knockout MSC exosomes, we confirmed that exosomal miR-21a-5p is transferred into myocardium and is a major cardioprotective paracrine factor produced by MSCs acting via synergistic activity on multiple pathways. The data supports that residual cardioprotective effect may be due to other ncRNA or protein cargo. In silico analyses support that MSC exosomes may also contribute to angiogenesis, cell proliferation and other aspects of cardiac repair. (hide) EV-METRIC 11% (25th 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 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 (d)(U)C Adj. k-factor 209.7 (pelleting) / 60.38 (washing) Protein markers EV: TSG101/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells mesenchymal stem cells EV-harvesting Medium EV-depleted serum Preparation of EDS 1.5h at 100,000g Cell viability 90 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 Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 209.7 Wash: time (min) 70 Wash: Rotor Type TLA-100.3 Wash: speed (g) 100000 Wash: adjusted k-factor 60.38 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins TSG101, CD9 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Amnis ImageStream Hardware adjustment Calibration bead size 100-150 EV concentration Yes Extra information Number of particles per microgram protein in final sample was 4.5 X 10E4

	EV200164	1/2	Homo sapiens	Serum	ExoQuick	Jiao, Chenwei	2017	0%
Study summary Full title Exosomal miR-34s panel as potential novel diagnostic and prognostic biomarker in patients with hepatoblastoma All authors Chenwei Jiao, Xiaohu Jiao, Anzhi Zhu, Juntao Ge, Xiaoqing Xu Journal J Pediatr Surg Abstract Purpose: The aim of this study is to identify the diagnostic values of serum exosomal miRNA-34s of (show more...) Purpose: The aim of this study is to identify the diagnostic values of serum exosomal miRNA-34s of patients with HB in a large Asian group and explore the prognostic value of the exosomal miRNA-34s panel compared with other risk factors. Methods: We retrospectively reviewed 89 children with HB. Among these patients, 63 patients were included as training group to build the diagnostic model for HB. 26 patients were defined as the validation group. The expressions of miRNA-34s were detected by real-time PCR. The comparison of diagnostic and prognostic performance of serum exosomal miRNA-34s was measured using the area under ROC curve (AUC). Results: For patients in the training group, expression of miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with control group in serum exosomes. Between HB training group and the control group, exosomal miRNA-34a, miRNA-34b and miRNA-34c had no significant differences compared with the AFP level in diagnosing HB. The performance of the exosomal miRNA-34s panel in differentiating the HB training group from the control group was superior to the AFP level. The value of the exosomal miRNA-34s panel in predicting prognosis of patients with HB was superior to other risk factors in both training group and validation group. Conclusions: In this study, we found that the expression of exosomal miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with the control group, and we confirmed the exosomal miRNA-34s panel could be defined as a diagnostic and prognostic biomarker for patients with HB. (hide) EV-METRIC 0% (median: 13% 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 Serum 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 ExoQuick Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Control condition Separation Method Commercial kit ExoQuick Protein Concentration Method Not determined Flow cytometry Hardware adjustments Characterization: Particle analysis

	EV200164	2/2	Homo sapiens	Serum	ExoQuick	Jiao, Chenwei	2017	0%
Study summary Full title Exosomal miR-34s panel as potential novel diagnostic and prognostic biomarker in patients with hepatoblastoma All authors Chenwei Jiao, Xiaohu Jiao, Anzhi Zhu, Juntao Ge, Xiaoqing Xu Journal J Pediatr Surg Abstract Purpose: The aim of this study is to identify the diagnostic values of serum exosomal miRNA-34s of (show more...) Purpose: The aim of this study is to identify the diagnostic values of serum exosomal miRNA-34s of patients with HB in a large Asian group and explore the prognostic value of the exosomal miRNA-34s panel compared with other risk factors. Methods: We retrospectively reviewed 89 children with HB. Among these patients, 63 patients were included as training group to build the diagnostic model for HB. 26 patients were defined as the validation group. The expressions of miRNA-34s were detected by real-time PCR. The comparison of diagnostic and prognostic performance of serum exosomal miRNA-34s was measured using the area under ROC curve (AUC). Results: For patients in the training group, expression of miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with control group in serum exosomes. Between HB training group and the control group, exosomal miRNA-34a, miRNA-34b and miRNA-34c had no significant differences compared with the AFP level in diagnosing HB. The performance of the exosomal miRNA-34s panel in differentiating the HB training group from the control group was superior to the AFP level. The value of the exosomal miRNA-34s panel in predicting prognosis of patients with HB was superior to other risk factors in both training group and validation group. Conclusions: In this study, we found that the expression of exosomal miRNA-34a, miRNA-34b and miRNA-34c was significantly lower in patients with HB compared with the control group, and we confirmed the exosomal miRNA-34s panel could be defined as a diagnostic and prognostic biomarker for patients with HB. (hide) EV-METRIC 0% (median: 13% 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 Serum Sample origin hepatoblastoma 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 ExoQuick Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition hepatoblastoma Separation Method Commercial kit ExoQuick Protein Concentration Method Not determined Flow cytometry Hardware adjustments Characterization: Particle analysis

	EV200105	1/3	Homo sapiens	Blood plasma	Macherey-Nagel Exosome Precipiation Solution	Orsolya Biró	2017	0%
Study summary Full title Various levels of circulating exosomal total-miRNA and miR-210 hypoxamiR in different forms of pregnancy hypertension All authors Orsolya Biró, Bálint Alasztics, Attila Molvarec, József Joó, Bálint Nagy, János Rigó Jr 2 Journal Pregnancy Hypertension Abstract Introduction: Hypertension is a common complication during pregnancy, affecting 10% of pregnant wome (show more...)Introduction: Hypertension is a common complication during pregnancy, affecting 10% of pregnant women worldwide. Several microRNA (miRNA) were shown to be involved in hypertensive disorders of pregnancy. In preeclampsia (PE), placental dysfunction causes the enhanced release of extracellular vesicle-derived miRNAs. The hypoxia-sensitive hsa-mir-210 is the most common PE-associated miRNA, but its exosomal profile has not been investigated. Objectives: Our aims were to measure exosomal total-miRNA concentration and to perform expression analysis of circulating exosomal hsa-miR-210 in women affected by chronic hypertension (CHT) gestational hypertension (GHT) or PE. Materials and methods: We collected plasma samples from women with CHT, GHT, PE (moderate: mPE and severe: sPE) and from normotensive pregnancies. Exosomal miRNAs were extracted and miRNA concentration was measured. RT-PCR was carried out with hsa-miR-210-3p-specific primers and relative expression was calculated using the comparative Ct method. Results: The total-miRNA concentration was different in the disease subgroups, and was significantly higher in mPE and sPE compared to the other groups. We found a significant difference in the relative exosomal hsa-miR-210-3p expression between all hypertensive groups compared to the normotensive samples, but significant upregulation was only observed in case of mPE and sPE patients. Both the level of total-miRNA and hsa-miR-210 expression was higher in case of severe PE. Conclusions: The level of circulating exosomal total-miRNA and hsa-miR-210 was elevated in women with PE, and it was higher in the severe form. We showed that hsa-miR-210 is secreted via exosomes, which may have a role in the pathomechanism of the disease. (hide) EV-METRIC 0% (median: 22% 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 Macherey-Nagel Exosome Precipiation Solution Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Healthy pregnant Separation Method Commercial kit Macherey-Nagel Exosome Precipiation Solution Protein Concentration Method Not determined Characterization: Particle analysis

	EV200105	2/3	Homo sapiens	Blood plasma	Macherey-Nagel Exosome Precipiation Solution	Orsolya Biró	2017	0%
Study summary Full title Various levels of circulating exosomal total-miRNA and miR-210 hypoxamiR in different forms of pregnancy hypertension All authors Orsolya Biró, Bálint Alasztics, Attila Molvarec, József Joó, Bálint Nagy, János Rigó Jr 2 Journal Pregnancy Hypertension Abstract Introduction: Hypertension is a common complication during pregnancy, affecting 10% of pregnant wome (show more...)Introduction: Hypertension is a common complication during pregnancy, affecting 10% of pregnant women worldwide. Several microRNA (miRNA) were shown to be involved in hypertensive disorders of pregnancy. In preeclampsia (PE), placental dysfunction causes the enhanced release of extracellular vesicle-derived miRNAs. The hypoxia-sensitive hsa-mir-210 is the most common PE-associated miRNA, but its exosomal profile has not been investigated. Objectives: Our aims were to measure exosomal total-miRNA concentration and to perform expression analysis of circulating exosomal hsa-miR-210 in women affected by chronic hypertension (CHT) gestational hypertension (GHT) or PE. Materials and methods: We collected plasma samples from women with CHT, GHT, PE (moderate: mPE and severe: sPE) and from normotensive pregnancies. Exosomal miRNAs were extracted and miRNA concentration was measured. RT-PCR was carried out with hsa-miR-210-3p-specific primers and relative expression was calculated using the comparative Ct method. Results: The total-miRNA concentration was different in the disease subgroups, and was significantly higher in mPE and sPE compared to the other groups. We found a significant difference in the relative exosomal hsa-miR-210-3p expression between all hypertensive groups compared to the normotensive samples, but significant upregulation was only observed in case of mPE and sPE patients. Both the level of total-miRNA and hsa-miR-210 expression was higher in case of severe PE. Conclusions: The level of circulating exosomal total-miRNA and hsa-miR-210 was elevated in women with PE, and it was higher in the severe form. We showed that hsa-miR-210 is secreted via exosomes, which may have a role in the pathomechanism of the disease. (hide) EV-METRIC 0% (median: 22% 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 Pre-eclampsia 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 Macherey-Nagel Exosome Precipiation Solution Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Pre-eclampsia Separation Method Commercial kit Macherey-Nagel Exosome Precipiation Solution Protein Concentration Method Not determined Characterization: Particle analysis

	EV200105	3/3	Homo sapiens	Blood plasma	Macherey-Nagel Exosome Precipiation Solution	Orsolya Biró	2017	0%
Study summary Full title Various levels of circulating exosomal total-miRNA and miR-210 hypoxamiR in different forms of pregnancy hypertension All authors Orsolya Biró, Bálint Alasztics, Attila Molvarec, József Joó, Bálint Nagy, János Rigó Jr 2 Journal Pregnancy Hypertension Abstract Introduction: Hypertension is a common complication during pregnancy, affecting 10% of pregnant wome (show more...)Introduction: Hypertension is a common complication during pregnancy, affecting 10% of pregnant women worldwide. Several microRNA (miRNA) were shown to be involved in hypertensive disorders of pregnancy. In preeclampsia (PE), placental dysfunction causes the enhanced release of extracellular vesicle-derived miRNAs. The hypoxia-sensitive hsa-mir-210 is the most common PE-associated miRNA, but its exosomal profile has not been investigated. Objectives: Our aims were to measure exosomal total-miRNA concentration and to perform expression analysis of circulating exosomal hsa-miR-210 in women affected by chronic hypertension (CHT) gestational hypertension (GHT) or PE. Materials and methods: We collected plasma samples from women with CHT, GHT, PE (moderate: mPE and severe: sPE) and from normotensive pregnancies. Exosomal miRNAs were extracted and miRNA concentration was measured. RT-PCR was carried out with hsa-miR-210-3p-specific primers and relative expression was calculated using the comparative Ct method. Results: The total-miRNA concentration was different in the disease subgroups, and was significantly higher in mPE and sPE compared to the other groups. We found a significant difference in the relative exosomal hsa-miR-210-3p expression between all hypertensive groups compared to the normotensive samples, but significant upregulation was only observed in case of mPE and sPE patients. Both the level of total-miRNA and hsa-miR-210 expression was higher in case of severe PE. Conclusions: The level of circulating exosomal total-miRNA and hsa-miR-210 was elevated in women with PE, and it was higher in the severe form. We showed that hsa-miR-210 is secreted via exosomes, which may have a role in the pathomechanism of the disease. (hide) EV-METRIC 0% (median: 22% 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 Hypertensive disorders (gestational and chronic) 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 Macherey-Nagel Exosome Precipiation Solution Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Hypertensive disorders (gestational and chronic) Separation Method Commercial kit Macherey-Nagel Exosome Precipiation Solution Protein Concentration Method Not determined Characterization: Particle analysis

	EV170071	1/1	Homo sapiens	Blood plasma	exoRNeasy	Del Re M	2017	0%
Study summary Full title The Detection of Androgen Receptor Splice Variant 7 in Plasma-derived Exosomal RNA Strongly Predicts Resistance to Hormonal Therapy in Metastatic Prostate Cancer Patients. All authors Del Re M, Biasco E, Crucitta S, Derosa L, Rofi E, Orlandini C, Miccoli M, Galli L, Falcone A, Jenster GW, van Schaik RH, Danesi R. Journal Eur Urol Abstract BACKGROUND: The androgen receptor splice variant 7 (AR-V7) is associated with resistance to hormonal (show more...)BACKGROUND: The androgen receptor splice variant 7 (AR-V7) is associated with resistance to hormonal therapy in castration-resistant prostate cancer (CRPC). Due to limitations of the methods available for AR-V7 analysis, the identification of a reliable detection method may facilitate the use of this biomarker in clinical practice. OBJECTIVE: To confirm AR-V7 as a predictor of resistance to hormonal therapy and develop a new approach to assess AR-V7 by highly sensitive digital droplet polymerase chain reaction (ddPCR) in plasma-derived exosomal RNA. DESIGN, SETTING, AND PARTICIPANTS: Plasma samples were collected from 36 CRPC patients before they began second-line hormonal treatment. Exosomes were isolated and RNA extracted for analysis of AR-V7 by ddPCR. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The absolute target gene concentration as copies per milliliter (copies/ml) was determined by ddPCR. Statistical analyses were performed with SPSS software (IBM Corp., Armonk, NY, USA). RESULTS AND LIMITATIONS: A total of 26 patients received abiraterone and 10 enzalutamide; 39% of patients were found to be AR-V7 positive (AR-V7+). Median progression-free survival was significantly longer in AR-V7 negative (AR-V7-) versus AR-V7+ patients (20 vs 3 mo; p<0.001). Overall survival was significantly shorter in AR-V7+ participants at baseline compared with AR-V7- participants (8 mo vs not reached; p<0.001). CONCLUSIONS: This study demonstrates that plasma-derived exosomal RNA is a reliable source of AR-V7 that can be detected sensitively by ddPCR assay. We also showed that resistance to hormonal therapy may be predicted by AR-V7, making it a clinically relevant biomarker. PATIENT SUMMARY: We report a first study on a method for androgen receptor splice variant 7 (AR-V7) detection in RNA extracted from cancer cell vesicles released in blood. Results confirmed the role of AR-V7 as a predictive biomarker of resistance to hormonal therapy. Our assay showed that vesicles are a reliable source of AR-V7 RNA and that the method is fast, highly sensitive, and affordable. (hide) EV-METRIC 0% (median: 22% 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 Metastatic Prostate Cancer Patients treated with enzalutamide or abiraterone Focus vesicles 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 exoRNeasy Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Metastatic Prostate Cancer Patients treated with enzalutamide or abiraterone Separation Method Commercial kit Other;exoRNeasy Protein Concentration Method Not determined Characterization: Particle analysis

	EV170069	1/3	Homo sapiens	Serum	ExoQuick	Kangas R	2017	0%
Study summary Full title Aging and serum exomiR content in women-effects of estrogenic hormone replacement therapy. All authors Kangas R, Törmäkangas T, Fey V, Pursiheimo J, Miinalainen I, Alen M, Kaprio J, Sipilä S, Säämänen AM, Kovanen V, Laakkonen EK. Journal Sci Rep Abstract Exosomes participate in intercellular messaging by transporting bioactive lipid-, protein- and RNA-m (show more...)Exosomes participate in intercellular messaging by transporting bioactive lipid-, protein- and RNA-molecules and -complexes. The contents of the exosomes reflect the physiological status of an individual making exosomes promising targets for biomarker analyses. In the present study we extracted exosome microRNAs (exomiRs) from serum samples of premenopausal women (n = 8) and monozygotic postmenopausal twins (n = 10 female pairs), discordant for the use of estrogenic hormone replacement therapy (HRT), in order to see whether the age or/and the use of HRT associates with exomiR content. A total of 241 exomiRs were detected by next generation sequencing, 10 showing age, 14 HRT and 10 age +HRT -related differences. When comparing the groups, differentially expressed miRs were predicted to affect cell proliferation processes showing inactivation with younger age and HRT usage. MiR-106-5p, -148a-3p, -27-3p, -126-5p, -28-3p and -30a-5p were significantly associated with serum 17β-estradiol. MiRs formed two hierarchical clusters being indicative of positive or negative health outcomes involving associations with body composition, serum 17β-estradiol, fat-, glucose- and inflammatory markers. Circulating exomiR clusters, obtained by NGS, could be used as indicators of metabolic and inflammatory status affected by hormonal changes at menopause. Furthermore, the individual effects of HRT-usage could be evaluated based on the serum exomiR signature. (hide) EV-METRIC 0% (median: 13% 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 Serum Sample origin premenopausal 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 ExoQuick Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Sample Condition premenopausal Separation Method Commercial kit ExoQuick Protein Concentration Method BCA Characterization: Particle analysis EM EM-type Report size (nm) EV concentration

	EV170069	2/3	Homo sapiens	Serum	ExoQuick	Kangas R	2017	0%
Study summary Full title Aging and serum exomiR content in women-effects of estrogenic hormone replacement therapy. All authors Kangas R, Törmäkangas T, Fey V, Pursiheimo J, Miinalainen I, Alen M, Kaprio J, Sipilä S, Säämänen AM, Kovanen V, Laakkonen EK. Journal Sci Rep Abstract Exosomes participate in intercellular messaging by transporting bioactive lipid-, protein- and RNA-m (show more...)Exosomes participate in intercellular messaging by transporting bioactive lipid-, protein- and RNA-molecules and -complexes. The contents of the exosomes reflect the physiological status of an individual making exosomes promising targets for biomarker analyses. In the present study we extracted exosome microRNAs (exomiRs) from serum samples of premenopausal women (n = 8) and monozygotic postmenopausal twins (n = 10 female pairs), discordant for the use of estrogenic hormone replacement therapy (HRT), in order to see whether the age or/and the use of HRT associates with exomiR content. A total of 241 exomiRs were detected by next generation sequencing, 10 showing age, 14 HRT and 10 age +HRT -related differences. When comparing the groups, differentially expressed miRs were predicted to affect cell proliferation processes showing inactivation with younger age and HRT usage. MiR-106-5p, -148a-3p, -27-3p, -126-5p, -28-3p and -30a-5p were significantly associated with serum 17β-estradiol. MiRs formed two hierarchical clusters being indicative of positive or negative health outcomes involving associations with body composition, serum 17β-estradiol, fat-, glucose- and inflammatory markers. Circulating exomiR clusters, obtained by NGS, could be used as indicators of metabolic and inflammatory status affected by hormonal changes at menopause. Furthermore, the individual effects of HRT-usage could be evaluated based on the serum exomiR signature. (hide) EV-METRIC 0% (median: 13% 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 Serum Sample origin Postmenopausal twin treated with estrogenic hormone replacement therapy 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 ExoQuick Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Sample Condition Postmenopausal twin treated with estrogenic hormone replacement therapy Separation Method Commercial kit ExoQuick Protein Concentration Method BCA EM EM-type Transmission-EM Image type Wide-field Report size (nm)

	EV170069	3/3	Homo sapiens	Serum	ExoQuick	Kangas R	2017	0%
Study summary Full title Aging and serum exomiR content in women-effects of estrogenic hormone replacement therapy. All authors Kangas R, Törmäkangas T, Fey V, Pursiheimo J, Miinalainen I, Alen M, Kaprio J, Sipilä S, Säämänen AM, Kovanen V, Laakkonen EK. Journal Sci Rep Abstract Exosomes participate in intercellular messaging by transporting bioactive lipid-, protein- and RNA-m (show more...)Exosomes participate in intercellular messaging by transporting bioactive lipid-, protein- and RNA-molecules and -complexes. The contents of the exosomes reflect the physiological status of an individual making exosomes promising targets for biomarker analyses. In the present study we extracted exosome microRNAs (exomiRs) from serum samples of premenopausal women (n = 8) and monozygotic postmenopausal twins (n = 10 female pairs), discordant for the use of estrogenic hormone replacement therapy (HRT), in order to see whether the age or/and the use of HRT associates with exomiR content. A total of 241 exomiRs were detected by next generation sequencing, 10 showing age, 14 HRT and 10 age +HRT -related differences. When comparing the groups, differentially expressed miRs were predicted to affect cell proliferation processes showing inactivation with younger age and HRT usage. MiR-106-5p, -148a-3p, -27-3p, -126-5p, -28-3p and -30a-5p were significantly associated with serum 17β-estradiol. MiRs formed two hierarchical clusters being indicative of positive or negative health outcomes involving associations with body composition, serum 17β-estradiol, fat-, glucose- and inflammatory markers. Circulating exomiR clusters, obtained by NGS, could be used as indicators of metabolic and inflammatory status affected by hormonal changes at menopause. Furthermore, the individual effects of HRT-usage could be evaluated based on the serum exomiR signature. (hide) EV-METRIC 0% (median: 13% 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 Serum Sample origin Postmenopausal twin not treated with estrogenic hormone replacement therapy 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 ExoQuick Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Sample Condition Postmenopausal twin not treated with estrogenic hormone replacement therapy Separation Method Commercial kit ExoQuick Protein Concentration Method BCA

	EV170068	1/1	Homo sapiens	Serum	(d)(U)C UF	Hao YX	2017	0%
Study summary Full title KRAS and BRAF mutations in serum exosomes from patients with colorectal cancer in a Chinese population. All authors Hao YX, Li YM, Ye M, Guo YY, Li QW, Peng XM, Wang Q, Zhang SF, Zhao HX, Zhang H, Li GH, Zhu JH, Xiao WH. Journal Oncol Lett Abstract The efficacy of epidermal growth factor receptor- targeted therapy is significantly associated with (show more...)The efficacy of epidermal growth factor receptor- targeted therapy is significantly associated with Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-raf serine/threonine kinase proto-oncogene (BRAF) mutation in patients with colorectal cancer (CRC), for which the standard gene testing is currently performed using tumor tissue DNA. The aim of the present study was to compare the presence of KRAS and BRAF mutations in the serum exosome and primary tumor tissue from patients with CRC. Genomic DNA were extracted from the tumor tissues of 35 patients with histologically-confirmed CRC and exosomal mRNA were obtained from peripheral blood, which were collected from the corresponding patients prior to surgery. Three mutations in the KRAS gene (codons 12, 13 and 61) and a mutation in the BRAF gene (codon 600) were detected using a polymerase chain reaction-based sequencing method and their presence were compared between tumor tissues and the matched serum exosomes. The KRAS mutation rates in tumor tissues and the matched serum exosomes were 57.6 and 42.4%, respectively, which was not significantly different (P=0.063). The detection rate of the BRAF mutation was 24.2 and 18.2% in tumor tissues and the matched serum exosomes, respectively, and there was no significant difference (P=0.500). The patients with CRC that had a KRAS mutation of codon 12 in exon 2 in their tumor tissues and serum exosomes were significantly older compared with those without this mutation (tumor tissue, P=0.002; serum exosome, P=0.022). The sensitivity of KRAS and BRAF mutation detection using exosomal mRNA was 73.7 and 75%, respectively. The specificity of the detected mutations exhibited an efficiency of 100%, and the total consistency rate was 94.9 and 93.9% for KRAS and BRAF mutations, respectively. These results suggested that serum exosomal mRNA may be used as a novel source for the rapid and non-invasive genotyping of patients with CRC. (hide) EV-METRIC 0% (median: 13% 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 Serum Sample origin colorectal cancer 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 (d)(U)C + UF Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Sample Condition colorectal cancer 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) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 120000 Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Protein Concentration Method Not determined Characterization: Particle analysis EM EM-type Transmission-EM/ Immuno-EM Proteïns CD63 Image type Wide-field Report size (nm) 30-80

	EV170066	1/5	Homo sapiens	Cell culture supernatant	ExoQuick	Zhang R	2017	0%
Study summary Full title Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in non-small cell lung cancer. All authors Zhang R, Xia Y, Wang Z, Zheng J, Chen Y, Li X, Wang Y, Ming H. Journal Biochem Biophys Res Commun Abstract Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), M (show more...)Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), MALAT-1 was first identified lncRNA that was related to lung cancer metastasis. However, the relationship between exosomal lncRNAs and the diagnosis and prognosis of NSCLC was poorly understood. The aim of this study is to evaluate the clinical significance of serum exosomal MALAT-1 as a biomarker in the metastasis of NSCLC. In this study, we firstly isolated the exosomes from healthy subjects and NSCLC patients. Then we measured the expression levels of MALAT-1 contained in exosomes, and found that exosomal MALAT-1 was highly expressed in NSCLC patients, more importantly, the levels of exosomal MALAT-1 were positively associated with tumor stage and lymphatic metastasis. In addition, we decreased MALAT-1 expression by short hairpin RNA and conducted a series of assays including MTT, cell cycle, colony formation, wound-healing scratch and Annexin/V PI by flow cytometry in human lung cancer cell lines. These in vitro studies demonstrated that serum exosome-derived long noncoding RNA MALAT-1 promoted the tumor growth and migration, and prevented tumor cells from apoptosis in lung cancer cell lines. Taken together, this study shed a light on utilizing MALAT-1 in exosomes as a non-invasive serum-based tumor biomarker for diagnosis and prognosis of NSCLC. (hide) EV-METRIC 0% (median: 22% 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 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 ExoQuick Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells A549 EV-harvesting Medium Not specified Separation Method Commercial kit ExoQuick Protein Concentration Method BCA Characterization: Particle analysis

	EV170066	2/5	Homo sapiens	Cell culture supernatant	ExoQuick	Zhang R	2017	0%
Study summary Full title Serum long non coding RNA MALAT-1 protected by exosomes is up-regulated and promotes cell proliferation and migration in non-small cell lung cancer. All authors Zhang R, Xia Y, Wang Z, Zheng J, Chen Y, Li X, Wang Y, Ming H. Journal Biochem Biophys Res Commun Abstract Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), M (show more...)Circulating lncRNAs have been defined as a novel biomarker for non-small cell lung cancer (NSCLC), MALAT-1 was first identified lncRNA that was related to lung cancer metastasis. However, the relationship between exosomal lncRNAs and the diagnosis and prognosis of NSCLC was poorly understood. The aim of this study is to evaluate the clinical significance of serum exosomal MALAT-1 as a biomarker in the metastasis of NSCLC. In this study, we firstly isolated the exosomes from healthy subjects and NSCLC patients. Then we measured the expression levels of MALAT-1 contained in exosomes, and found that exosomal MALAT-1 was highly expressed in NSCLC patients, more importantly, the levels of exosomal MALAT-1 were positively associated with tumor stage and lymphatic metastasis. In addition, we decreased MALAT-1 expression by short hairpin RNA and conducted a series of assays including MTT, cell cycle, colony formation, wound-healing scratch and Annexin/V PI by flow cytometry in human lung cancer cell lines. These in vitro studies demonstrated that serum exosome-derived long noncoding RNA MALAT-1 promoted the tumor growth and migration, and prevented tumor cells from apoptosis in lung cancer cell lines. Taken together, this study shed a light on utilizing MALAT-1 in exosomes as a non-invasive serum-based tumor biomarker for diagnosis and prognosis of NSCLC. (hide) EV-METRIC 0% (median: 22% 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 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 ExoQuick Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells H1299 EV-harvesting Medium

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