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You searched for: EV210163 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210163 1/5 Homo sapiens Cell culture supernatant (d)(U)C/ SEC (non-commercial)/ UF/ Filtration Shepherd Megan 2021 67%

Study summary

Full title
All authors
Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon
Journal
Cell Commun Signal
Abstract
Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide)
EV-METRIC
67% (91st 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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Ultrafiltration
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Decidual
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
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: rotor type
Type 70.1Ti
Pelleting: speed (g)
100 000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
0,1
Sample volume/column (mL)
0,1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ ANIXA S/ iCAM/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
CD81/ Flotillin1/ ANIXA S/ iCAM/ TSG101/ CD63/ CD9/ Alix
Detected contaminants
GM130
Not detected contaminants
GM130
Proteomics database
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
115
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 2.00e+0
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up
Report size (nm)
100
EV210163 2/5 Homo sapiens Cell culture supernatant (d)(U)C/ SEC (non-commercial)/ UF/ Filtration Shepherd Megan 2021 67%

Study summary

Full title
All authors
Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon
Journal
Cell Commun Signal
Abstract
Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide)
EV-METRIC
67% (91st 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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Ultrafiltration
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Myometrial
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
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: rotor type
Type 70.1Ti
Pelleting: speed (g)
100 000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
0,1
Sample volume/column (mL)
0,1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ ANIXA S/ iCAM/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
CD81/ Flotillin1/ ANIXA S/ iCAM/ TSG101/ CD63/ CD9/ Alix
Detected contaminants
GM130
Proteomics database
No
Detected EV-associated proteins
CD81/ CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
116,075
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 2.00e+0
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
100
EV210163 3/5 Homo sapiens Cell culture supernatant (d)(U)C/ SEC (non-commercial)/ UF/ Filtration Shepherd Megan 2021 67%

Study summary

Full title
All authors
Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon
Journal
Cell Commun Signal
Abstract
Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide)
EV-METRIC
67% (91st 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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Oxidative stress inducer (CSE) treated
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Ultrafiltration
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Decidual
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
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: rotor type
Type 70.1Ti
Pelleting: speed (g)
100 000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
0,1
Sample volume/column (mL)
0,1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ Alix/ CD9/ CD63/ TSG101/ ANIXA S/ iCAM/ CD81
Detected contaminants
GM130
Proteomics database
No
Detected EV-associated proteins
CD81/ CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
116
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 2.00e+0
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
100
EV210163 4/5 Homo sapiens Cell culture supernatant (d)(U)C/ SEC (non-commercial)/ UF/ Filtration Shepherd Megan 2021 67%

Study summary

Full title
All authors
Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon
Journal
Cell Commun Signal
Abstract
Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide)
EV-METRIC
67% (91st 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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Oxidative stress inducer (CSE) treated
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Ultrafiltration
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Myometrial
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
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: rotor type
Type 70.1Ti
Pelleting: speed (g)
100 000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
0,1
Sample volume/column (mL)
0,1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ Alix/ ANIXA S/ iCAM/ CD9/ CD63/ TSG101/ CD81
Detected contaminants
GM130
Proteomics database
No
Detected EV-associated proteins
CD81/ CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
116
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 2.00e+0
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
90
EV210163 5/5 Homo sapiens Cell culture supernatant (d)(U)C/ SEC (non-commercial)/ UF/ Filtration Shepherd Megan 2021 67%

Study summary

Full title
All authors
Megan C Shepherd, Enkhtuya Radnaa, Ourlad Alzeus Tantengco, Talar Kechichian, Rheanna Urrabaz-Garza, Ananth Kumar Kammala, Samantha Sheller-Miller, Ramkumar Menon
Journal
Cell Commun Signal
Abstract
Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channe (show more...)Background: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. Methods: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. Results: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. Conclusion: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract. (hide)
EV-METRIC
67% (91st 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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Inflammation inducer-TNF-alpha treated
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Ultrafiltration
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ iCAM/ Flotillin1/ ANIXA S/ CD9
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Myometrial
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
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: rotor type
Type 70.1Ti
Pelleting: speed (g)
100 000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
0,1
Sample volume/column (mL)
0,1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ ANIXA S/ iCAM/ CD9/ CD63/ TSG101/ Alix/ CD81
Detected contaminants
GM130
Proteomics database
No
Detected EV-associated proteins
CD81/ CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
120
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 2.00e+0
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
90
1 - 5 of 5
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV210163
species
Homo sapiens
sample type
Cell culture
cell type
Decidual
Myometrial
Decidual
Myometrial
Myometrial
condition
Control condition
Control condition
Oxidative
stress inducer (CSE) treated
Oxidative
stress inducer (CSE) treated
Inflammation
inducer-TNF-alpha treated
separation protocol
dUC/ Size-exclusion
chromatography (non-commercial)/
Ultrafiltration/ Filtration
dUC/ Size-exclusion
chromatography (non-commercial)/
Ultrafiltration/ Filtration
dUC/ Size-exclusion
chromatography (non-commercial)/
Ultrafiltration/ Filtration
dUC/ Size-exclusion
chromatography (non-commercial)/
Ultrafiltration/ Filtration
dUC/ Size-exclusion
chromatography (non-commercial)/
Ultrafiltration/ Filtration
Exp. nr.
1
2
3
4
5
EV-METRIC %
67
67
67
67
67