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You searched for: EV200085 (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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV200085 1/5 Rattus norvegicus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Rohit Kumar 2020 45%

Study summary

Full title
All authors
Rohit Kumar, Qilin Tang, Stephan A Müller, Pan Gao, Diana Mahlstedt, Sofia Zampagni, Yi Tan, Andreas Klingl, Kai Bötzel, Stefan F Lichtenthaler, Günter U Höglinger, Thomas Koeglsperger
Journal
Advanced Science
Abstract
Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecul (show more...)Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch-clamp electrophysiology and pH-sensitive dye imaging is used to examine stimulus-evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high-frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus-evoked MVB-PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking-down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB-PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release. (hide)
EV-METRIC
45% (82nd 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
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
CD63-pHluorin transduced
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
Density gradient + (Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD81/ Flotillin1/ CD9/ GFP/ VAMP3/ VAMP2
non-EV: None
Proteomics
yes
EV density (g/ml)
1.08-1.14
Show all info
Study aim
Function/Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
CD63-pHluorin transduced
EV-producing cells
Primary Hippocampus neurons
EV-harvesting Medium
Serum free
Cell viability
Yes
Cell number specification
Yes
Separation Method
(Differential) (ultra)centrifugation
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)
90
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
10%
Highest density fraction
30%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
350000
Duration (min)
60
Fraction volume (mL)
0.49
Fraction processing
Centrifugation
Pelleting: volume per fraction
3
Pelleting: duration (min)
30
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ Flotillin1/ CD9/ GFP/ VAMP3
Not detected EV-associated proteins
VAMP2
Proteomics database
Yes
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
0-500
EV concentration
Yes
EV200085 2/5 Rattus norvegicus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Rohit Kumar 2020 23%

Study summary

Full title
All authors
Rohit Kumar, Qilin Tang, Stephan A Müller, Pan Gao, Diana Mahlstedt, Sofia Zampagni, Yi Tan, Andreas Klingl, Kai Bötzel, Stefan F Lichtenthaler, Günter U Höglinger, Thomas Koeglsperger
Journal
Advanced Science
Abstract
Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecul (show more...)Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch-clamp electrophysiology and pH-sensitive dye imaging is used to examine stimulus-evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high-frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus-evoked MVB-PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking-down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB-PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release. (hide)
EV-METRIC
23% (53rd 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
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
CD63-pHluorin transduced + bFGF 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
Density gradient + (Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD81/ Flotillin1/ CD9/ GFP/ VAMP3
non-EV: VAMP2
Proteomics
yes
Show all info
Study aim
Function/Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
CD63-pHluorin transduced + bFGF treated
EV-producing cells
Primary Hippocampus neurons
EV-harvesting Medium
Serum free
Cell viability
Yes
Cell number specification
Yes
Separation Method
(Differential) (ultra)centrifugation
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)
90
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ Flotillin1/ CD9/ GFP/ VAMP3
Not detected EV-associated proteins
VAMP2
Proteomics database
Yes
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
0-500
EV concentration
Yes
EV200085 3/5 Rattus norvegicus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Rohit Kumar 2020 23%

Study summary

Full title
All authors
Rohit Kumar, Qilin Tang, Stephan A Müller, Pan Gao, Diana Mahlstedt, Sofia Zampagni, Yi Tan, Andreas Klingl, Kai Bötzel, Stefan F Lichtenthaler, Günter U Höglinger, Thomas Koeglsperger
Journal
Advanced Science
Abstract
Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecul (show more...)Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch-clamp electrophysiology and pH-sensitive dye imaging is used to examine stimulus-evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high-frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus-evoked MVB-PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking-down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB-PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release. (hide)
EV-METRIC
23% (53rd 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
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
CD63-pHluorin transduced + siRNA VAMP3 + bFGF 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
Density gradient + (Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD81/ Flotillin1/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
CD63-pHluorin transduced + siRNA VAMP3 + bFGF treated
EV-producing cells
Primary Hippocampus neurons
EV-harvesting Medium
Serum free
Cell viability
Yes
Cell number specification
Yes
Separation Method
(Differential) (ultra)centrifugation
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)
90
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ Flotillin1/ CD9
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
0-500
EV concentration
Yes
EV200085 4/5 Rattus norvegicus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Rohit Kumar 2020 23%

Study summary

Full title
All authors
Rohit Kumar, Qilin Tang, Stephan A Müller, Pan Gao, Diana Mahlstedt, Sofia Zampagni, Yi Tan, Andreas Klingl, Kai Bötzel, Stefan F Lichtenthaler, Günter U Höglinger, Thomas Koeglsperger
Journal
Advanced Science
Abstract
Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecul (show more...)Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch-clamp electrophysiology and pH-sensitive dye imaging is used to examine stimulus-evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high-frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus-evoked MVB-PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking-down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB-PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release. (hide)
EV-METRIC
23% (53rd 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
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
CD63-pHluorin transduced + bFGF and BABPTA-AM 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
Density gradient + (Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD81/ GFP
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
CD63-pHluorin transduced + bFGF and BABPTA-AM treated
EV-producing cells
Primary Hippocampus neurons
EV-harvesting Medium
Serum free
Cell viability
Yes
Cell number specification
Yes
Separation Method
(Differential) (ultra)centrifugation
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)
90
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD81/ GFP
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
0-500
EV concentration
Yes
EV200085 5/5 Rattus norvegicus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Rohit Kumar 2020 15%

Study summary

Full title
All authors
Rohit Kumar, Qilin Tang, Stephan A Müller, Pan Gao, Diana Mahlstedt, Sofia Zampagni, Yi Tan, Andreas Klingl, Kai Bötzel, Stefan F Lichtenthaler, Günter U Höglinger, Thomas Koeglsperger
Journal
Advanced Science
Abstract
Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecul (show more...)Extracellular vesicles (EVs) are endogenous membrane-derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch-clamp electrophysiology and pH-sensitive dye imaging is used to examine stimulus-evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high-frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus-evoked MVB-PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking-down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB-PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release. (hide)
EV-METRIC
15% (41st 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
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
CD63-pHluorin transduced + bFGF and Genistein 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
Density gradient + (Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
CD63-pHluorin transduced + bFGF and Genistein treated
EV-producing cells
Primary Hippocampus neurons
EV-harvesting Medium
Serum free
Cell viability
Yes
Cell number specification
Yes
Separation Method
(Differential) (ultra)centrifugation
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)
90
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Protein Concentration Method
BCA
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
0-500
EV concentration
Yes
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