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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 Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles 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% (97th 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 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 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 Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles 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% (97th 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 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 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 Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles 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% (86th 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 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 Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles 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% (86th 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 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 Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles 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% (78th 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 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 Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles 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% (34th 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 Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles 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% (34th 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

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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

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