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You searched for: EV200049 (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
EV200049 1/2 Homo sapiens placental tissue culture supernatrant DG
(d)(U)C
Bergamelli, Mathilde 2021 100%

Study summary

Full title
All authors
Mathilde Bergamelli, Hélène Martin, Mélinda Bénard, Jérôme Ausseil, Jean-Michel Mansuy, Ilse Hurbain, Maïlys Mouysset, Marion Groussolles, Géraldine Cartron, Yann Tanguy le Gac, Nathalie Moinard, Elsa Suberbielle, Jacques Izopet, Charlotte Tscherning, Graça Raposo, Daniel Gonzalez-Dunia, Gisela D'Angelo, Cécile E Malnou
Journal
Front Cell Dev Biol
Abstract
Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of p (show more...)Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking. (hide)
EV-METRIC
100% (50th 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
placental tissue culture supernatrant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
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
DG
(d)(U)C
Protein markers
EV: CD29/ CD63/ CD81/ CD44/ CD326/ CD9
non-EV: Calreticulin
Proteomics
no
EV density (g/ml)
1.103
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
placental tissue culture supernatrant
Separation Method
(Differential) (ultra)centrifugation
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: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
25
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected EV-associated proteins
CD81/ CD9
Not detected contaminants
Calreticulin
Detected EV-associated proteins
CD9/ CD29/ CD44/ CD326/ CD81/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
143
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.00E+08
Particle analysis: flow cytometry
Flow cytometer type
mascquant VYB
Hardware adjustment
Use of calibration beads FITC positive, of different size (500, 240, 200 and 160 nm) and granulosity that allow bead population separation and calibration of the cytometer , creation of a gate on 200 nm and smaller events
Calibration bead size
0.16
EV concentration
Yes
EM
EM-type
Immuno-EM/ Transmission-EM
Proteïns
CD63
Image type
Close-up, Wide-field
Report size (nm)
95
EV200049 2/2 Homo sapiens placental tissue culture supernatrant DG
(d)(U)C
Bergamelli, Mathilde 2021 100%

Study summary

Full title
All authors
Mathilde Bergamelli, Hélène Martin, Mélinda Bénard, Jérôme Ausseil, Jean-Michel Mansuy, Ilse Hurbain, Maïlys Mouysset, Marion Groussolles, Géraldine Cartron, Yann Tanguy le Gac, Nathalie Moinard, Elsa Suberbielle, Jacques Izopet, Charlotte Tscherning, Graça Raposo, Daniel Gonzalez-Dunia, Gisela D'Angelo, Cécile E Malnou
Journal
Front Cell Dev Biol
Abstract
Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of p (show more...)Extracellular vesicles (EVs) have increasingly been recognized as key players in a wide variety of physiological and pathological contexts, including during pregnancy. Notably, EVs appear both as possible biomarkers and as mediators involved in the communication of the placenta with the maternal and fetal sides. A better understanding of the physiological and pathological roles of EVs strongly depends on the development of adequate and reliable study models, specifically at the beginning of pregnancy where many adverse pregnancy outcomes have their origin. In this study, we describe the isolation of small EVs from a histoculture model of first trimester placental explants in normal conditions as well as upon infection by human cytomegalovirus. Using bead-based multiplex cytometry and electron microscopy combined with biochemical approaches, we characterized these small EVs and defined their associated markers and ultrastructure. We observed that infection led to changes in the expression level of several surface markers, without affecting the secretion and integrity of small EVs. Our findings lay the foundation for studying the functional role of EVs during early pregnancy, along with the identification of new predictive biomarkers for the severity and outcome of this congenital infection, which are still sorely lacking. (hide)
EV-METRIC
100% (50th 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
placental tissue culture supernatrant
Sample origin
in vitro hCMV infected placental tissue
Focus vesicles
Other / small extracellular vesicles
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
DG
(d)(U)C
Protein markers
EV: CD29/ CD63/ CD81/ CD44/ CD326/ CD9
non-EV: Calreticulin
Proteomics
no
EV density (g/ml)
1.103
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
placental tissue culture supernatrant
Separation Method
(Differential) (ultra)centrifugation
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: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
25
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected EV-associated proteins
CD63/ CD9
Not detected contaminants
Calreticulin
Detected EV-associated proteins
CD9/ CD29/ CD44/ CD326/ CD81/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
Particle analysis: flow cytometry
Flow cytometer type
mascquant VYB
Hardware adjustment
Use of calibration beads FITC positive, of different size (500, 240, 200 and 160 nm) and granulosity that allow bead population separation and calibration of the cytometer , creation of a gate on 200 nm and smaller events
Calibration bead size
0.16
Report type
Mean
Reported size (nm)
140
EV concentration
Yes
EM
EM-type
Immuno-EM/ Transmission-EM
Proteïns
CD63
Image type
Close-up, Wide-field
Report size (nm)
100
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200049
species
Homo sapiens
sample type
placental
tissue culture supernatrant
condition
Control condition
in
vitro hCMV infected placental tissue
separation protocol
DG
(d)(U)C
DG
(d)(U)C
Exp. nr.
1
2
EV-METRIC %
100
100