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Results list Most used isolation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV170036	11/12	Homo sapiens	Serum	dUC	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% (66th 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles EV12 Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Origin Prostate cancer Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g 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	dUC	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% (46th 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles EV12 Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Origin Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g 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

	EV170035	1/4	Neisseria meningitidis	Cell culture supernatant	dUC	Vestad B	2017	14%
Study summary Full title Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. All authors Vestad B, Llorente A, Neurauter A, Phuyal S, Kierulf B, Kierulf P, Skotland T, Sandvig K, Haug KBF, Øvstebø R Journal J Extracell Vesicles Abstract Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in (show more...)Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted. (hide) EV-METRIC 14% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles Outer membrane vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Neisseria meningitidis Sample Type Cell culture supernatant EV-producing cells Neisseria meningitidis EV-harvesting Medium Not specified Origin Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: speed (g) 140000 Protein Concentration Method Not determined Characterization: Particle analysis NTA Report type Mean,Mode Reported size (nm) Mean: 138-156;Mode: 102-122 EV concentration Yes Particle yield 5.8E8-8.9E8 EM EM-type Transmission-EM Image type Wide-field Extra information This was a technical report and a variation study focusing on NTA, detailed characterization of EVs less important. NTA data of these EVs not obtained using instrument-optimized settings.

	EV170035	2/4	Homo sapiens	Cell culture supernatant	dUC	Vestad B	2017	14%
Study summary Full title Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. All authors Vestad B, Llorente A, Neurauter A, Phuyal S, Kierulf B, Kierulf P, Skotland T, Sandvig K, Haug KBF, Øvstebø R Journal J Extracell Vesicles Abstract Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in (show more...)Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted. (hide) EV-METRIC 14% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles (shedding) microvesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary monocytes EV-harvesting Medium EV-depleted serum Origin Stimulated with E. coli LPS Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 30 Pelleting: speed (g) 17000 Protein Concentration Method Not determined Characterization: Particle analysis NTA Report type Mean,Mode Reported size (nm) Mean: 167-171;Mode: 119-127 EV concentration Yes Particle yield 3.9E8-4.3E8 EM EM-type Transmission-EM Image type Wide-field Extra information This was a technical report and a variation study focusing on NTA, detailed characterization of EVs less important. NTA data of these EVs obtained using instrument-optimized settings.

	EV170035	3/4	Homo sapiens	Cell culture supernatant	dUC	Vestad B	2017	14%
Study summary Full title Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. All authors Vestad B, Llorente A, Neurauter A, Phuyal S, Kierulf B, Kierulf P, Skotland T, Sandvig K, Haug KBF, Øvstebø R Journal J Extracell Vesicles Abstract Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in (show more...)Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted. (hide) EV-METRIC 14% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles exosome Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: CD63 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium Serum free medium Origin Control condition Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 Protein Concentration Method Not determined Characterization: Particle analysis NTA Report type Mean,Mode Reported size (nm) Mean:125-135;Mode:89-92 EV concentration Yes Particle yield 2.9E8-3.1E8 EM EM-type Immune-EM Image type Wide-field Extra information This was a technical report and a variation study focusing on NTA, detailed characterization of EVs less important. NTA data of these EVs obtained using instrument-optimized settings.

	EV170022	1/5	Streptococcus pneumoniae	Cell culture supernatant	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + 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 EV-producing cells Streptococcus pneumoniae EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + 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 EV-producing cells Moraxella catarrhalis EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + 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 EV-producing cells non-typeable Haemophilus influenzae EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration + SEC + UF Protein markers EV: CD63/ CD81 non-EV: None Proteomics no Show all info Study aim Function, Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + 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 EV-producing cells Pseudomonas aeruginosa EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Cell viability 95 Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + 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 EV-producing cells non-typeable Haemophilus influenzae EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration + SEC + UF Protein markers EV: CD63/ CD81 non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + 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 EV-producing cells Streptococcus pneumoniae EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + 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 EV-producing cells Pseudomonas aeruginosa EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (43rd 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles membrane vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + 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 EV-producing cells Moraxella catarrhalis EV-harvesting Medium EV-depleted serum Origin Control condition Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: 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	dUC 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% (44th 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + SEC + UF Protein markers EV: CD9/ CD63 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Urine Origin Control condition Isolation Method Differential ultracentrifugation dUC: 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

	EV170000	2/2	Homo sapiens	Serum	dUC Filtration	Thomou T	2017	0%
Study summary Full title Adipose-derived circulating miRNAs regulate gene expression in other tissues. All authors Thomou T, Mori MA, Dreyfuss JM, Konishi M, Sakaguchi M, Wolfrum C, Rao TN, Winnay JN, Garcia-Martin R, Grinspoon SK, Gorden P, Kahn Journal Nature Abstract Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism thro (show more...)Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue-brown especially-into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3' UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine. (hide) EV-METRIC 0% (median: 11% 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 Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles exosome Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: speed (g) 100000 Wash: time (min) 60 Wash: speed (g) 100000 Characterization: Particle analysis Other particle analysis name(1) EXOCET ELISA assay EV-concentration Yes

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