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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
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
Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
  • Experiments differ in Sample type, Vesicle type
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Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
  • Experiments differ in Sample type, Vesicle type
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Experiment number
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Experiment number
  • Experiments differ in Particle analysis
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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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