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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220058	1/5	Homo sapiens	HCT-116	(d)(U)C	Lee CH	2018	44%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 44% (84th 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ HSP70/ TSPAN1/ LGALS3BP/ SLC1A/ CLDN7/ GPRC5A/ CD63 non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT-116 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 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 CD63/ TSPAN1/ LGALS3BP/ SLC1A/ CLDN7/ GPRC5A/ HSP70/ Alix Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-500 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV220058	2/5	Homo sapiens	HT-29	(d)(U)C	Lee CH	2018	44%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 44% (84th 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 (Differential) (ultra)centrifugation Protein markers EV: Alix/ HSP70/ TSPAN1/ LGALS3BP/ SLC1A/ CLDN7/ GPRC5A/ CD63 non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT-29 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 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 CD63/ TSPAN1/ LGALS3BP/ SLC1A/ CLDN7/ GPRC5A/ HSP70/ Alix Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-500 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV220058	4/5	Homo sapiens	Blood plasma	ExoQuick	Lee CH	2018	38%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Protein markers EV: TSPAN1/ CD63/ CD9/ TSPAN1/ LGALS3BP/ SLC1A5/ CLDN7/ GPRC5A non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit 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 TSPAN1/ LGALS3BP/ SLC1A5/ CLDN7/ GPRC5A/ CD9 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ TSPAN1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220058	5/5	Homo sapiens	Blood plasma	ExoQuick	Lee CH	2018	38%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 38% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Colon cancer 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 Commercial method Protein markers EV: TSPAN1/ CD63/ CD9/ TSPAN1/ LGALS3BP/ SLC1A5/ CLDN7/ GPRC5A non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit 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 TSPAN1/ LGALS3BP/ SLC1A5/ CLDN7/ GPRC5A/ CD9 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ TSPAN1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220058	3/5	Homo sapiens	CRL-1541	(d)(U)C	Lee CH	2018	33%
Study summary Full title Discovery of a diagnostic biomarker for colon cancer through proteomic profiling of small extracellular vesicles. All authors Lee CH, Im EJ, Moon PG, Baek MC Journal BMC Cancer Abstract Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information reg (show more...)Small extracellular vesicles (small-EVs) are membranous vesicles that contain unique information regarding the condition of cells and contribute to the recruitment and reprogramming of components associated with the tumor environment. Therefore, many researchers have suggested that small-EV proteins are potential biomarkers for diseases such as cancer. Colon cancer (CC) is one of the most common causes of cancer-related deaths worldwide. Biomarkers such as carcinoembryonic antigen (CEA) show low sensitivity (~ 40%), and thus the demand for novel biomarkers for CC diagnosis is increasing. (hide) EV-METRIC 33% (75th 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 (Differential) (ultra)centrifugation Protein markers EV: CLDN7/ GPRC5A/ HSP70/ TSPAN1/ LGALS3BP/ SLC1A5 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CRL-1541 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: speed (g) 100000 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 HSP70/ CLDN7/ GPRC5A Not detected EV-associated proteins TSPAN1/ LGALS3BP/ SLC1A5 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV220058
species	Homo sapiens
sample type	Cell culture	Cell culture	Blood plasma	Blood plasma	Cell culture
cell type	HCT-116	HT-29	NA	NA	CRL-1541
medium	EV-depleted medium	EV-depleted medium	NA	NA	EV-depleted medium
condition	Control condition	Control condition	Control condition	Colon cancer	Control condition
separation protocol	dUC	dUC	ExoQuick	ExoQuick	dUC
Exp. nr.	1	2	4	5	3
EV-METRIC %	44	44	38	38	33