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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210322	1/5	Homo sapiens	MDAMB231	(d)(U)C	Kibria G	2016	44%
Study summary Full title A rapid, automated surface protein profiling of single circulating exosomes in human blood. All authors Kibria G, Ramos EK, Lee KE, Bedoyan S, Huang S, Samaeekia R, Athman JJ, Harding CV, Lötvall J, Harris L, Thompson CL, Liu H Journal Sci Rep Abstract Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human di (show more...)Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids. (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 exosome 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: CD63/ beta-actin/ CD44/ LAMP2B/ CD47 non-EV: Grp94 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 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 SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ beta-actin/ CD44/ LAMP2B Detected contaminants Grp94 Flow cytometry Type of Flow cytometry Apogee A50 Micro Flow Cytometer Calibration bead size 0.11 Detected EV-associated proteins CD44/ CD63/ CD47 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 89 EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100

	EV210322	3/5	Homo sapiens	Serum	(d)(U)C	Kibria G	2016	44%
Study summary Full title A rapid, automated surface protein profiling of single circulating exosomes in human blood. All authors Kibria G, Ramos EK, Lee KE, Bedoyan S, Huang S, Samaeekia R, Athman JJ, Harding CV, Lötvall J, Harris L, Thompson CL, Liu H Journal Sci Rep Abstract Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human di (show more...)Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids. (hide) EV-METRIC 44% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles exosome 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: CD63/ CD81/ LAMP2B/ beta-actin non-EV: Grp94 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 TLA 50.1 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 2 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD81/ LAMP2B/ beta-actin Detected contaminants Grp94 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100

	EV210322	4/5	Homo sapiens	Serum	(d)(U)C Total Exosome Isolation	Kibria G	2016	33%
Study summary Full title A rapid, automated surface protein profiling of single circulating exosomes in human blood. All authors Kibria G, Ramos EK, Lee KE, Bedoyan S, Huang S, Samaeekia R, Athman JJ, Harding CV, Lötvall J, Harris L, Thompson CL, Liu H Journal Sci Rep Abstract Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human di (show more...)Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids. (hide) EV-METRIC 33% (76th 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 Serum Sample origin Control condition Focus vesicles exosome 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 Total Exosome Isolation Protein markers EV: CD63/ CD81/ LAMP2B/ beta-actin/ CD47/ CD44 non-EV: Grp94 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 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 TLA 50.1 Pelleting: speed (g) 100000 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ CD81/ LAMP2B/ beta-actin Detected contaminants Grp94 ELISA Detected EV-associated proteins CD47 Flow cytometry Type of Flow cytometry Apogee A50 Micro Flow Cytometer Calibration bead size 0.11 Detected EV-associated proteins CD47/ CD44 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100

	EV210322	2/5	Homo sapiens	MCF12A	(d)(U)C	Kibria G	2016	22%
Study summary Full title A rapid, automated surface protein profiling of single circulating exosomes in human blood. All authors Kibria G, Ramos EK, Lee KE, Bedoyan S, Huang S, Samaeekia R, Athman JJ, Harding CV, Lötvall J, Harris L, Thompson CL, Liu H Journal Sci Rep Abstract Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human di (show more...)Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids. (hide) EV-METRIC 22% (58th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome 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: beta-actin/ CD44 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF12A EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 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 SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins beta-actin Not detected EV-associated proteins CD44 Flow cytometry Type of Flow cytometry Apogee A50 Micro Flow Cytometer Calibration bead size 0.11 Detected EV-associated proteins CD44 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210322	5/5	Homo sapiens	Serum	(d)(U)C Total Exosome Isolation	Kibria G	2016	11%
Study summary Full title A rapid, automated surface protein profiling of single circulating exosomes in human blood. All authors Kibria G, Ramos EK, Lee KE, Bedoyan S, Huang S, Samaeekia R, Athman JJ, Harding CV, Lötvall J, Harris L, Thompson CL, Liu H Journal Sci Rep Abstract Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human di (show more...)Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids. (hide) EV-METRIC 11% (40th 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 Serum Sample origin Breast cancer Focus vesicles exosome 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 Total Exosome Isolation Protein markers EV: CD47/ CD44 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 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 TLA 50.1 Pelleting: speed (g) 100000 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined ELISA Detected EV-associated proteins CD47 Flow cytometry Type of Flow cytometry Apogee A50 Micro Flow Cytometer Calibration bead size 0.11 Detected EV-associated proteins CD47/ CD44 Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210322
species	Homo sapiens
sample type	Cell culture	Serum	Serum	Cell culture	Serum
cell type	MDAMB231	NA	NA	MCF12A	NA
medium	EV-depleted medium	NA	NA	EV-depleted medium	NA
condition	Control condition	Control condition	Control condition	Control condition	Breast cancer
separation protocol	dUC	dUC	dUC/ Total Exosome Isolation	dUC	dUC/ Total Exosome Isolation
Exp. nr.	1	3	4	2	5
EV-METRIC %	44	44	33	22	11