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You searched for: EV210154 (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 ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210154 1/2 Homo sapiens human invasive proliferative extravillous cytotrophoblast (HIPEC) DG
(d)(U)C
Bergamelli M 2022 100%

Study summary

Full title
All authors
Bergamelli M, Martin H, Aubert Y, Mansuy JM, Marcellin M, Burlet-Schiltz O, Hurbain I, Raposo G, Izopet J, Fournier T, Benchoua A, Bénard M, Groussolles M, Cartron G, Tanguy Le Gac Y, Moinard N, D'Angelo G, Malnou CE
Journal
Viruses
Abstract
Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pre (show more...)Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection. (hide)
EV-METRIC
100% (99th 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
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ Alix/ CD63/ CD9/ CD81
non-EV: calnexin/ TOM20
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human invasive proliferative extravillous cytotrophoblast (HIPEC)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell count
1,00E+08
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 performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: 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
30
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
30
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
CD63
Not detected contaminants
calnexin/ TOM20
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
160
EV concentration
Yes
Particle yield
as number of particles per million cells: 1,00E+06
Particle analysis: flow cytometry
Flow cytometer type
Mascquant VYB Myltenyi
Hardware adjustment
Mascquant VYB Myltenyi set up fluorescent beads SSC Megamix, with calibration on gates of 160nm 200nm 250nm and 500nm
Calibration bead size
0.16/ 0.2/ 0.25/ 0.5
EV concentration
Yes
Particle yield
as number of particles per million cells: 1,00E+06
EM
EM-type
Immuno-EM/ Transmission-EM
EM protein
CD9/ CD81/ CD63
Image type
Close-up, Wide-field
Report size (nm)
120
EV210154 2/2 Homo sapiens human invasive proliferative extravillous cytotrophoblast (HIPEC) DG
(d)(U)C
Bergamelli M 2022 100%

Study summary

Full title
All authors
Bergamelli M, Martin H, Aubert Y, Mansuy JM, Marcellin M, Burlet-Schiltz O, Hurbain I, Raposo G, Izopet J, Fournier T, Benchoua A, Bénard M, Groussolles M, Cartron G, Tanguy Le Gac Y, Moinard N, D'Angelo G, Malnou CE
Journal
Viruses
Abstract
Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pre (show more...)Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection. (hide)
EV-METRIC
100% (99th 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
infected by Human Cytomegalovirus / clinical strain VHL/E
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ Alix/ CD63/ CD9/ CD81
non-EV: calnexin/ TOM20
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human invasive proliferative extravillous cytotrophoblast (HIPEC)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell count
1,00E+08
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 performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: 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
30
Pelleting: duration (min)
60
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
30
Pelleting-wash: duration (min)
60
Pelleting-wash: speed (g)
SW 32 Ti
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix/ CD81
Not detected contaminants
calnexin/ TOM20
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
as number of particles per million cells: 1,00E+06
Particle analysis: flow cytometry
Flow cytometer type
Macsquant VYB Myltenyi
Hardware adjustment
Mascquant VYB Myltenyi set up fluorescent beads SSC Megamix, with calibration on gates of 160nm 200nm 250nm and 500nm
Calibration bead size
0.16/ 0.2/ 0.25/ 0.5
Report type
Mean
Reported size (nm)
160
EV concentration
Yes
Particle yield
as number of particles per million cells: 1,00E+06
EM
EM-type
Immuno-EM/ Transmission-EM
EM protein
CD9/ CD81/ CD63
Image type
Close-up, Wide-field
Report size (nm)
110
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV210154
species
Homo sapiens
sample type
Cell culture
cell type
human invasive
proliferative extravillous
cytotrophoblast (HIPEC)
condition
Control condition
infected by
Human
Cytomegalovirus / clinical
strain VHL/E
separation protocol
Density
gradient/ dUC
Density
gradient/ dUC
Exp. nr.
1
2
EV-METRIC %
100
100