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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180071	1/3	Homo sapiens	Blood plasma	SEC UF	Brahmer A	2019	63%
Study summary Full title Platelets, endothelial cells and leukocytes contribute to the exercise-triggered release of extracellular vesicles into the circulation. All authors Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM. Journal J Extracell Vesicles Abstract Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide) EV-METRIC 63% (91st 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 Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture SEC UF Protein markers EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142 non-EV: / ApoA1 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ CD9/ TSG101/ Syntenin/ CD41b/ CD63 Not detected EV-associated proteins Sarcoglycan-alpha Detected contaminants ApoA1 Not detected contaminants Detected EV-associated proteins CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 106 EM EM-type Transmission-EM Image type Wide-field

	EV180071	2/3	Homo sapiens	Blood plasma	SEC UF	Brahmer A	2019	50%
Study summary Full title Platelets, endothelial cells and leukocytes contribute to the exercise-triggered release of extracellular vesicles into the circulation. All authors Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM. Journal J Extracell Vesicles Abstract Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide) EV-METRIC 50% (83rd 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 Blood plasma Sample origin RQ 0.9 during exercise Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture SEC UF Protein markers EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142 non-EV: / ApoA1 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101/ CD9/ Syntenin/ CD41b/ CD63 Not detected EV-associated proteins Sarcoglycan-alpha Detected contaminants ApoA1 Not detected contaminants Detected EV-associated proteins CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported

	EV180071	3/3	Homo sapiens	Blood plasma	SEC UF	Brahmer A	2019	50%
Study summary Full title Platelets, endothelial cells and leukocytes contribute to the exercise-triggered release of extracellular vesicles into the circulation. All authors Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM. Journal J Extracell Vesicles Abstract Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide) EV-METRIC 50% (83rd 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 Blood plasma Sample origin post exercise Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture SEC UF Protein markers EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142 non-EV: / ApoA1 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 30 Membrane type Regenerated cellulose Commercial kit Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101/ CD9/ Syntenin/ CD41b/ CD63 Not detected EV-associated proteins Sarcoglycan-alpha Detected contaminants ApoA1 Not detected contaminants Detected EV-associated proteins CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported

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EV-TRACK ID	EV180071
species	Homo sapiens
sample type	Blood plasma
condition	Control condition	RQ 0.9 during exercise	post exercise
separation protocol	SEC UF	SEC UF	SEC UF
Exp. nr.	1	2	3
EV-METRIC %	63	50	50