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You searched for: EV200015 (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
EV200015 1/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Streck, Nicholas 2020 89%

Study summary

Full title
All authors
Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich
Journal
J Virol
Abstract
Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide)
EV-METRIC
89% (98th 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
extracellular vesicle
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
Filtration
Protein markers
EV: TSG101/ CD81/ CD63
non-EV: Tubulin
Proteomics
no
EV density (g/ml)
Density not calculated
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary human dermal fibroblasts
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 performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
130000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
5%
Highest density fraction
41%
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
130000
Duration (min)
960
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
120
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
130000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
PMID previous EV protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ TSG101/ CD81
Not detected contaminants
Tubulin
Characterization: Lipid analysis
No
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
170
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.01E+10
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report type
Not Reported
EV-concentration
No
EV200015 2/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Streck, Nicholas 2020 89%

Study summary

Full title
All authors
Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich
Journal
J Virol
Abstract
Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide)
EV-METRIC
89% (98th 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
HCMV infected 72hpi
Focus vesicles
extracellular vesicle
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
Filtration
Protein markers
EV: TSG101/ CD81/ HCMV glycoprotein B/ CD63
non-EV: Tubulin
Proteomics
no
EV density (g/ml)
Density not calculated
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary human dermal fibroblasts
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 performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
130000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
5%
Highest density fraction
41%
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
130000
Duration (min)
960
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
120
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
130000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
PMID previous EV protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ HCMV glycoprotein B/ TSG101/ CD81
Not detected contaminants
Tubulin
Characterization: Lipid analysis
No
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
158
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 3.51E+10
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report type
Not Reported
EV-concentration
No
EV200015 3/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Streck, Nicholas 2020 44%

Study summary

Full title
All authors
Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich
Journal
J Virol
Abstract
Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide)
EV-METRIC
44% (76th 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
HCMV infected 72hpi DMSO-treated
Focus vesicles
extracellular vesicle
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
Filtration
Protein markers
EV: CD63
non-EV: None
Proteomics
no
EV density (g/ml)
Density not calculated
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary human dermal fibroblasts
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 performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
130000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
5%
Highest density fraction
41%
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
130000
Duration (min)
960
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
120
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
130000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
PMID previous EV protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Not Reported
Particle yield
NA NA
Report type
Not Reported
EV-concentration
No
EV200015 4/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Streck, Nicholas 2020 44%

Study summary

Full title
All authors
Nicholas T Streck, Yuanjun Zhao, Jeffrey M Sundstrom, Nicholas J Buchkovich
Journal
J Virol
Abstract
Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways withi (show more...)Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus that leads to serious health consequences in neonatal or immunocompromised patients. Clinical management of infection in these at-risk groups remains a serious concern even with approved antiviral therapies available. It is necessary to increase our understanding of the cellular changes that occur during infection and their importance to virus spread. This may help to identify new targets during infection that will lead to the development of novel treatment strategies. Extracellular vesicles (EVs) represent an important method of intercellular communication in the human host. This study finds that HCMV manipulates this pathway to increase the efficiency of virus spread to uninfected cells. This finding defines a new layer of host manipulation induced by HCMV infection that leads to enhanced virus spread. (hide)
EV-METRIC
44% (76th 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
HCMV infected 72hpi GW4869-treated
Focus vesicles
extracellular vesicle
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
Filtration
Protein markers
EV: CD63
non-EV: None
Proteomics
no
EV density (g/ml)
Density not calculated
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary human dermal fibroblasts
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 performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
130000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
5%
Highest density fraction
41%
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
130000
Duration (min)
960
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
120
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
130000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
PMID previous EV protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample Not reported
Report type
Not Reported
EV-concentration
No
1 - 4 of 4
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200015
species
Homo sapiens
sample type
Cell culture
cell type
primary
human dermal fibroblasts
condition
Control condition
HCMV infected 72hpi
HCMV
infected 72hpi DMSO-treated
HCMV
infected 72hpi GW4869-treated
separation protocol
DG
(d)(U)C
Filtration
DG
(d)(U)C
Filtration
DG
(d)(U)C
Filtration
DG
(d)(U)C
Filtration
Exp. nr.
1
2
3
4
EV-METRIC %
89
89
44
44