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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210297	1/7	Mus musculus	M1	qEV IAF PEG precipitation	Nørgård, Mikkel	2022	50%
Study summary Full title A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles All authors Mikkel Ø. Nørgård, Lasse B. Steffensen, Didde R. Hansen, Ernst-Martin Füchtbauer, Morten B. Engelund, Henrik Dimke, Ditte C. Andersen & Per Svenningsen Journal Sci Rep Abstract The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since (show more...)The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function. (hide) EV-METRIC 50% (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 CD9truc-EGFP expressing 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 qEV Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: Alix/ Flotillin-1/ EGFP/ TSG101 non-EV: Actin/ Lamin A/C Proteomics no Show all info Study aim New methodological development Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells M1 EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) EGFP Other Name other separation method qEV Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ Flotillin-1/ EGFP/ TSG101 Not detected contaminants Actin/ Lamin A/C Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 30-150

	EV210297	2/7	Mus musculus	Blood plasma	IAF PEG precipitation	Nørgård, Mikkel	2022	25%
Study summary Full title A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles All authors Mikkel Ø. Nørgård, Lasse B. Steffensen, Didde R. Hansen, Ernst-Martin Füchtbauer, Morten B. Engelund, Henrik Dimke, Ditte C. Andersen & Per Svenningsen Journal Sci Rep Abstract The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since (show more...)The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function. (hide) EV-METRIC 25% (58th 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 CD9truc-EGFPxCMV-Cre 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: Alix/ EGFP non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Mus musculus Sample Type Blood plasma Separation Method Immunoaffinity capture Selected surface protein(s) EGFP Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins EGFP Not detected EV-associated proteins Alix Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210297	3/7	Mus musculus	Blood plasma	IAF PEG precipitation	Nørgård, Mikkel	2022	25%
Study summary Full title A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles All authors Mikkel Ø. Nørgård, Lasse B. Steffensen, Didde R. Hansen, Ernst-Martin Füchtbauer, Morten B. Engelund, Henrik Dimke, Ditte C. Andersen & Per Svenningsen Journal Sci Rep Abstract The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since (show more...)The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function. (hide) EV-METRIC 25% (58th 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 CD9truc-EGFPxPax8-Cre 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: Alix/ EGFP non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Mus musculus Sample Type Blood plasma Separation Method Immunoaffinity capture Selected surface protein(s) EGFP Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Not detected EV-associated proteins Alix/ EGFP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210297	4/7	Mus musculus	Blood plasma	IAF PEG precipitation	Nørgård, Mikkel	2022	25%
Study summary Full title A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles All authors Mikkel Ø. Nørgård, Lasse B. Steffensen, Didde R. Hansen, Ernst-Martin Füchtbauer, Morten B. Engelund, Henrik Dimke, Ditte C. Andersen & Per Svenningsen Journal Sci Rep Abstract The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since (show more...)The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function. (hide) EV-METRIC 25% (58th 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 CD9truc-EGFPx?MHC-Cre 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: Alix/ EGFP non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Mus musculus Sample Type Blood plasma Separation Method Immunoaffinity capture Selected surface protein(s) EGFP Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ EGFP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210297	5/7	Mus musculus	urine	IAF PEG precipitation	Nørgård, Mikkel	2022	25%
Study summary Full title A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles All authors Mikkel Ø. Nørgård, Lasse B. Steffensen, Didde R. Hansen, Ernst-Martin Füchtbauer, Morten B. Engelund, Henrik Dimke, Ditte C. Andersen & Per Svenningsen Journal Sci Rep Abstract The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since (show more...)The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function. (hide) EV-METRIC 25% (55th 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 urine Sample origin CD9truc-EGFPxCMV-Cre 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: CD81 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Mus musculus Sample Type urine Separation Method Immunoaffinity capture Selected surface protein(s) EGFP Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210297	6/7	Mus musculus	urine	IAF PEG precipitation	Nørgård, Mikkel	2022	25%
Study summary Full title A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles All authors Mikkel Ø. Nørgård, Lasse B. Steffensen, Didde R. Hansen, Ernst-Martin Füchtbauer, Morten B. Engelund, Henrik Dimke, Ditte C. Andersen & Per Svenningsen Journal Sci Rep Abstract The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since (show more...)The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function. (hide) EV-METRIC 25% (55th 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 urine Sample origin CD9truc-EGFPxPax8-Cre 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: CD81/ EGFP non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Mus musculus Sample Type urine Separation Method Immunoaffinity capture Selected surface protein(s) EGFP Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81/ EGFP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210297	7/7	Mus musculus	urine	IAF PEG precipitation	Nørgård, Mikkel	2022	25%
Study summary Full title A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles All authors Mikkel Ø. Nørgård, Lasse B. Steffensen, Didde R. Hansen, Ernst-Martin Füchtbauer, Morten B. Engelund, Henrik Dimke, Ditte C. Andersen & Per Svenningsen Journal Sci Rep Abstract The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since (show more...)The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function. (hide) EV-METRIC 25% (55th 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 urine Sample origin CD9truc-EGFPx?MHC-Cre 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: CD81/ EGFP non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Mus musculus Sample Type urine Separation Method Immunoaffinity capture Selected surface protein(s) EGFP Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Not detected EV-associated proteins CD81/ EGFP Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210297
species	Mus musculus
sample type	Cell culture	Blood plasma	Blood plasma	Blood plasma	urine	urine	urine
cell type	M1	NA	NA	NA	NA	NA	NA
medium	Serum free medium	NA	NA	NA	NA	NA	NA
condition	CD9truc-EGFP expressing	CD9truc-EGFPxCMV-Cre	CD9truc-EGFPxPax8-Cre	CD9truc-EGFPx?MHC-Cre	CD9truc-EGFPxCMV-Cre	CD9truc-EGFPxPax8-Cre	CD9truc-EGFPx?MHC-Cre
separation protocol	qEV/ IAF capture (non-commercial)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation
Exp. nr.	1	2	3	4	5	6	7
EV-METRIC %	50	25	25	25	25	25	25

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