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You searched for: EV160009 (EV-TRACK ID)

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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 condition
Experiment number
  • Experiments differ in Sample condition
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV160009 1/2 Homo sapiens Cell culture supernatant dUC
dUC
Filtration
Benedikter BJ 2016 44%

Study summary

Full title
All authors
Benedikter BJ, Volgers C, van Eijck PH, Wouters EFM, Savelkoul PHM, Reynaert NL, Haenen GRMM, Rohde GGU, Weseler AR, Stassen FRM
Journal
J Cell Sci
Abstract
INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) wi (show more...)INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
extracellular vesicle / 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 + dUC + Filtration
Adj. k-factor
133.2 (pelleting)
Protein markers
EV: CD63
non-EV: grp94
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Exposed to cigarette smoke extract
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
75
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
117734
Pelleting: adjusted k-factor
133.2
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected contaminants
grp94
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
60-300
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
50-160
EV160009 2/2 Homo sapiens Cell culture supernatant dUC
dUC
Filtration
Benedikter BJ 2016 44%

Study summary

Full title
All authors
Benedikter BJ, Volgers C, van Eijck PH, Wouters EFM, Savelkoul PHM, Reynaert NL, Haenen GRMM, Rohde GGU, Weseler AR, Stassen FRM
Journal
J Cell Sci
Abstract
INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) wi (show more...)INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. (hide)
EV-METRIC
44% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
extracellular vesicle / 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 + dUC + Filtration
Adj. k-factor
133.2 (pelleting)
Protein markers
EV: CD63
non-EV: grp94
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability
100
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
117734
Pelleting: adjusted k-factor
133.2
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected contaminants
grp94
Fluorescent NTA
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
60-300
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
50-160
1 - 2 of 2
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV160009
species
Homo sapiens
sample type
Cell culture
cell type
BEAS2B
condition
Exposed
to cigarette smoke extract
Control condition
isolation protocol
dUC
dUC
Filtration
dUC
dUC
Filtration
case number
1
2
EV-METRIC %
44
44