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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200001	1/9	Homo sapiens	EBC1	DG (d)(U)C	Useckaite, Zivile	2020	89%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 89% (99th 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 DG (d)(U)C Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells EBC1 EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 99 Cell count 2.25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Density gradient Type Continuous Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 1080 Fraction volume (mL) 5 Fraction processing Centrifugation Pelleting: volume per fraction 17;17mL Pelleting: duration (min) 120 Pelleting: rotor type SW 32.1 Ti;SW 32 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ Syntenin1/ Actinin4 Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 111 EV concentration Yes Particle yield Yes, as number of particles per million cells 86300000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	4/9	Homo sapiens	H596	DG (d)(U)C	Useckaite, Zivile	2020	89%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 89% (99th 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 DG (d)(U)C Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H596 EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 98 Cell count 2.03E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Density gradient Type Continuous Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 1080 Fraction volume (mL) 5 Fraction processing Centrifugation Pelleting: volume per fraction 17;17mL Pelleting: duration (min) 120 Pelleting: rotor type SW 32.1 Ti;SW 32 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Syntenin1/ Actinin4/ CD9/ CD63 Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 106 EV concentration Yes Particle yield Yes, as number of particles per million cells 89500000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	7/9	Homo sapiens	Hs746T	DG (d)(U)C	Useckaite, Zivile	2020	89%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 89% (99th 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 DG (d)(U)C Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Hs746T EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 98 Cell count 2.03E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Density gradient Type Continuous Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 1080 Fraction volume (mL) 5 Fraction processing Centrifugation Pelleting: volume per fraction 17;17mL Pelleting: duration (min) 120 Pelleting: rotor type SW 32.1 Ti;SW 32 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Syntenin1/ Actinin4/ CD9/ CD63 Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 107 EV concentration Yes Particle yield Yes, as number of particles per million cells 29000000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	2/9	Homo sapiens	EBC1	(d)(U)C	Useckaite, Zivile	2020	67%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 67% (94th 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: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells EBC1 EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 99 Cell count 2.25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ Syntenin1/ Actinin4 Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 158 EV concentration Yes Particle yield Yes, as number of particles per million cells 20800000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	3/9	Homo sapiens	EBC1	(d)(U)C ExoQuick	Useckaite, Zivile	2020	67%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 67% (94th 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 ExoQuick Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ GRP94/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells EBC1 EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 99 Cell count 2.25E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Other Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ Syntenin1/ GRP94 Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 119 EV concentration Yes Particle yield Yes, as number of particles per million cells 771000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	5/9	Homo sapiens	H596	(d)(U)C	Useckaite, Zivile	2020	67%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 67% (94th 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: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H596 EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 98 Cell count 2.03E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Syntenin1/ Actinin4/ CD9/ CD63 Detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 146 EV concentration Yes Particle yield Yes, as number of particles per million cells 11400000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	6/9	Homo sapiens	H596	(d)(U)C ExoQuick	Useckaite, Zivile	2020	67%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 67% (94th 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 ExoQuick Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H596 EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 98 Cell count 2.03E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Syntenin1/ Actinin4/ CD9/ CD63 Detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 123 EV concentration Yes Particle yield Yes, as number of particles per million cells 5710000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	8/9	Homo sapiens	Hs746T	(d)(U)C	Useckaite, Zivile	2020	67%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 67% (94th 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: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Hs746T EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 98 Cell count 2.03E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Syntenin1/ Actinin4/ CD9/ CD63 Detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 147 EV concentration Yes Particle yield Yes, as number of particles per million cells 11600000000 EM EM-type Transmission-EM Image type Close-up

	EV200001	9/9	Homo sapiens	Hs746T	(d)(U)C ExoQuick	Useckaite, Zivile	2020	67%
Study summary Full title Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation All authors Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll Journal Sci Rep Abstract MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide) EV-METRIC 67% (94th 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 ExoQuick Protein markers EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ GRP94/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Hs746T EV-harvesting Medium EV-depleted medium Preparation of EDS 18h, 120000g;Other preparation Cell viability (%) 98 Cell count 2.03E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 10000 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Syntenin1/ GRP94/ CD9/ CD63 Not detected contaminants GRP94 ELISA Antibody details provided? No Detected EV-associated proteins MET/ pMET (pY1234/1235) Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Hardware adaptation to ~100nm EV's Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n Calibration bead size 80+ Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLA-DR Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 111 EV concentration Yes Particle yield Yes, as number of particles per million cells 3740000000 EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV200001
species	Homo sapiens
sample type	Cell culture
cell type	EBC1	H596	Hs746T	EBC1	EBC1	H596	H596	Hs746T	Hs746T
condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition
separation protocol	DG (d)(U)C	DG (d)(U)C	DG (d)(U)C	(d)(U)C	(d)(U)C ExoQuick	(d)(U)C	(d)(U)C ExoQuick	(d)(U)C	(d)(U)C ExoQuick
Exp. nr.	1	4	7	2	3	5	6	8	9
EV-METRIC %	89	89	89	67	67	67	67	67	67

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