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Results list Most used separation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV200085	1/5	Rattus norvegicus	Cell culture supernatant	DG (d)(U)C	Rohit Kumar	2020	45%
Study summary Full title Fibroblast Growth Factor 2-Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons 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% (79th 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 Cell Name Primary Hippocampus neurons 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C 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 ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes 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 Proteomics database Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes

	EV200085	2/5	Rattus norvegicus	Cell culture supernatant	DG (d)(U)C	Rohit Kumar	2020	23%
Study summary Full title Fibroblast Growth Factor 2-Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons 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% (49th 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 Cell Name Primary Hippocampus neurons 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C 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 ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes 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 Proteomics database Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes

	EV200085	3/5	Rattus norvegicus	Cell culture supernatant	DG (d)(U)C	Rohit Kumar	2020	23%
Study summary Full title Fibroblast Growth Factor 2-Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons 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% (49th 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 Cell Name Primary Hippocampus neurons 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C 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 ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes 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 NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes

	EV200085	4/5	Rattus norvegicus	Cell culture supernatant	DG (d)(U)C	Rohit Kumar	2020	23%
Study summary Full title Fibroblast Growth Factor 2-Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons 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% (49th 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 Cell Name Primary Hippocampus neurons 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C 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 ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes 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 NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes

	EV200085	5/5	Rattus norvegicus	Cell culture supernatant	DG (d)(U)C	Rohit Kumar	2020	15%
Study summary Full title Fibroblast Growth Factor 2-Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons 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% (39th 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 Cell Name Primary Hippocampus neurons 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C 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 ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes 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 NTA Report type Size range/distribution Reported size (nm) 0-500 EV concentration Yes

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