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You searched for: EV210202 (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
EV210202 1/7 Mus musculus 3T3-L1 DG
(d)(U)C
Durcin, Maëva 2017 78%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
78% (96th 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
Large 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: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.14-1.24
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
3T3-L1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
13000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
13000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
31
Lowest density fraction
0.4M
Highest density fraction
2M
Total gradient volume, incl. sample (mL)
12000
Sample volume (mL)
500
Orientation
Top-down
Rotor type
Not specified
Speed (g)
200000
Duration (min)
1080
Fraction volume (mL)
1000
Fraction processing
Centrifugation
Pelleting: volume per fraction
6000
Pelleting: duration (min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Mfge8/ caveolin-1/ b-actin/ Flotillin2/ TSG101/ Alix
Not detected EV-associated proteins
CD81
Flow cytometry
Type of Flow cytometry
MACSQuant flow cytometer
Calibration bead size
4µm
Antibody details provided?
No
Detected EV-associated proteins
Annexin V
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
160
EV concentration
Yes
Particle yield
EV secreted per adipocyte;Yes, other: 11
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-100
EV210202 2/7 Mus musculus 3T3-L1 DG
(d)(U)C
Durcin, Maëva 2017 78%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
78% (96th 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
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: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.14-1.20
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
3T3-L1
EV-harvesting Medium
Serum free medium
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: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
31
Lowest density fraction
0.4M
Highest density fraction
2M
Total gradient volume, incl. sample (mL)
12000
Sample volume (mL)
500
Orientation
Top-down
Rotor type
Not specified
Speed (g)
200000
Duration (min)
1080
Fraction volume (mL)
1000
Fraction processing
Centrifugation
Pelleting: volume per fraction
6000
Pelleting: duration (min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Mfge8/ Flotillin2/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
caveolin-1/ b-actin
Flow cytometry
Type of Flow cytometry
MACSQuant flow cytometer
Calibration bead size
4µm
Antibody details provided?
No
Not detected EV-associated proteins
Annexin V
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
100
EV concentration
Yes
Particle yield
EV secreted per adipocyte;Yes, other: 843
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
90-800
EV210202 6/7 Mus musculus Primary adipocytes (d)(U)C Durcin, Maëva 2017 45%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
45% (80th 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
Large 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
(d)(U)C
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
Primary adipocytes
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
13000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
13000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Mfge8/ Flotillin2/ caveolin-1
Not detected EV-associated proteins
CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
Reported size (nm)
110-150
EV concentration
Yes
Particle yield
EV sercreted per adipocyte;Yes, other: 100
EM
EM-type
Transmission-EM
Image type
Close-up
EV210202 7/7 Mus musculus Primary adipocytes (d)(U)C Durcin, Maëva 2017 45%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
45% (80th 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
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
(d)(U)C
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
Primary adipocytes
EV-harvesting Medium
Serum free medium
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: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Mfge8/ Flotillin2/ caveolin-1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
Reported size (nm)
80-100
EV concentration
Yes
Particle yield
EV sercreted per adipocyte;Yes, other: 200
EM
EM-type
Transmission-EM
Image type
Close-up
EV210202 3/7 Mus musculus 3T3-L1 DG
(d)(U)C
Durcin, Maëva 2017 34%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
34% (68th 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
Total 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: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
3T3-L1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Mfge8/ Flotillin2/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
caveolin-1/ b-actin
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV210202 4/7 Mus musculus 3T3-L1 (d)(U)C Durcin, Maëva 2017 13%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
13% (32nd 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
OA/PA/8Br cAMP/Iono/AngII/INFgamma/TNFalpha-treated
Focus vesicles
Large 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
(d)(U)C
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
3T3-L1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
13000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
13000
Characterization: Protein analysis
None
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
EV secreted per adipocyte;Yes, other: OA: 15/PA: 85/8Br cAMP: 15/Iono: 40/AngII: 15/INFgamma: 15/TNFalpha: 25
EV210202 5/7 Mus musculus 3T3-L1 (d)(U)C Durcin, Maëva 2017 13%

Study summary

Full title
All authors
Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide)
EV-METRIC
13% (32nd 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
OA/PA/8Br cAMP/Iono/AngII/INFgamma/TNFalpha-treated
Focus vesicles
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
(d)(U)C
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
3T3-L1
EV-harvesting Medium
Serum free medium
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: time(min)
60
Pelleting: rotor type
MLA-50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
60
Wash: Rotor Type
MLA-50
Wash: speed (g)
100000
Characterization: Protein analysis
None
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
EV secreted per adipocyte;Yes, other: OA: 600/PA: 1500/8Br cAMP: 500/Iono: 900/AngII: 500/INFgamma: 500/TNFalpha: 1200
1 - 7 of 7
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV210202
species
Mus
musculus
sample type
Cell
culture
cell type
3T3-L1
3T3-L1
Primary
adipocytes
Primary
adipocytes
3T3-L1
3T3-L1
3T3-L1
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
OA/PA/8Br
cAMP/Iono/AngII/INFgamma/TNFalpha-treated
OA/PA/8Br
cAMP/Iono/AngII/INFgamma/TNFalpha-treated
separation protocol
DG
(d)(U)C
DG
(d)(U)C
(d)(U)C
(d)(U)C
DG
(d)(U)C
(d)(U)C
(d)(U)C
vesicle related term
Large
EVs
Small
EVs
Large
EVs
Small
EVs
Total
EVs
Large
EVs
Small
EVs
Exp. nr.
1
2
6
7
3
4
5
EV-METRIC %
78
78
45
45
34
13
13