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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210126	1/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (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 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD63/ GFP/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 71 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	2/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (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 CD47-GFP 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD10 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD63/ GFP/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 75 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	3/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (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 TSPAN14-GFP 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD11 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD63/ TSG101/ GFP/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 73 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	4/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (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 CD63-GFP 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD12 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ GFP/ CD63/ TSG101/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 74 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	5/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (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 CD81TM-GFP 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD13 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 74 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	6/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (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 SDCBP-GFP 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD14 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 76 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	7/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (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 APMAP-GFP 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD15 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 76 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	8/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	78%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (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 CD63TM-GFP 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 (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD16 non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD9 Detected contaminants calnexin Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Detected EV-associated proteins CD81/ CD9/ CD63/ GFP Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type NanoAnalyzer N30 (nanoFCM) Hardware adjustment Calibration bead size 0.068; 0.091; 0.113; 0.155 Report type Mean Reported size (nm) 73 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210126	9/9	Homo sapiens	Expi293F	(d)(U)C	Silva, Andreia;Lázaro-Ibáñez, Elisa	2021	11%
Study summary Full title Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution All authors Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker Journal J Extracell Vesicles Abstract Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide) EV-METRIC 11% (30th 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 GFP 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 (d)(U)C Protein markers EV: None non-EV: calnexin Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 80 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 100 Wash: time (min) 120 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Flow cytometry Type of Flow cytometry NanoAnalyzer N30 (nanoFCM) Calibration bead size 0.068; 0.091; 0.113; 0.155 Detected EV-associated proteins GFP Characterization: Lipid analysis No

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EV-TRACK ID	EV210126
species	Homo sapiens
sample type	Cell culture
cell type	Expi293F
condition	Control condition	CD47-GFP	TSPAN14-GFP	CD63-GFP	CD81TM-GFP	SDCBP-GFP	APMAP-GFP	CD63TM-GFP	GFP
separation protocol	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C	(d)(U)C
Exp. nr.	1	2	3	4	5	6	7	8	9
EV-METRIC %	78	78	78	78	78	78	78	78	11

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