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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180026	5/7	Homo sapiens	Serum	(d)(U)C	Wenzhe Li	2018	77%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 77% (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 Serum 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 Adj. k-factor 120.7 (pelleting) / 120.7 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ HER2/ EpCAM non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Serum 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: time(min) 1200 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 130000 Pelleting: adjusted k-factor 120.7 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 130000 Wash: adjusted k-factor 120.7 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 30-120 Western Blot Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Albumin, Calnexin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 110.2 ± 12.95 nm NTA Report type Size range/distribution Reported size (nm) 124.4 ± 53.3 nm EV concentration Yes Particle yield 3290000000000 EM EM-type Transmission-EM/ Immune-EM EM protein EpCAM,HER2 Image type Close-up, Wide-field EV concentration Yes

	EV180026	1/7	Homo sapiens	SKBR3	(d)(U)C Filtration	Wenzhe Li	2018	55%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 55% (88th 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 Filtration Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ HER2/ EpCAM non-EV: Calnexin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SKBR3 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63, CD81, Flotillin-1, EpCAM, HER2 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 110.2 ± 12.95 NTA Report type Size range/distribution Reported size (nm) 124.4 ± 53.3 EM EM-type Transmission-EM/ Immune-EM EM protein EpCAM,HER2 Image type Close-up, Wide-field EV concentration Yes Extra information Particle/protein ratio was determined

	EV180026	3/7	Homo sapiens	Serum	(d)(U)C Filtration	Wenzhe Li	2018	55%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 55% (91st 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 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 Filtration Adj. k-factor 104.6 (pelleting) / 104.6 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ HER2/ EpCAM non-EV: Albumin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Serum 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: time(min) 1200 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Pelleting: adjusted k-factor 104.6 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Wash: adjusted k-factor 104.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 134.8 ± 52 EM EM-type Transmission-EM Image type Close-up, Wide-field EV concentration Yes Extra information Particle/protein ratio was determined

	EV180026	7/7	Homo sapiens	MCF7	(d)(U)C Filtration	Wenzhe Li	2018	44%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (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 (d)(U)C Filtration Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ EpCAM non-EV: Calnexin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63, CD81, Flotillin-1, EpCAM Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 141.5 ± 15.04 NTA Report type Size range/distribution Reported size (nm) 146.2 ± 60.2 EM EM-type Transmission-EM Image type Close-up EV concentration Yes

	EV180026	2/7	Homo sapiens	MCF10A	(d)(U)C Filtration	Wenzhe Li	2018	33%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 33% (74th 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 Filtration Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF10A EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No

	EV180026	4/7	Homo sapiens	MDAMB468	(d)(U)C Filtration	Wenzhe Li	2018	33%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 33% (74th 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 Filtration Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ EpCAM non-EV: Calnexin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB468 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63, CD81, Flotillin-1, EpCAM Not detected contaminants Calnexin Characterization: Lipid analysis No

	EV180026	6/7	Homo sapiens	Serum	(d)(U)C Filtration	Wenzhe Li	2018	33%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (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 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 Filtration Adj. k-factor 104.6 (pelleting) / 104.6 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ EpCAM non-EV: Albumin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Serum 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: time(min) 1200 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Pelleting: adjusted k-factor 104.6 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Wash: adjusted k-factor 104.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Albumin Characterization: Lipid analysis No

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EV-TRACK ID	EV180026
species	Homo sapiens
sample type	Serum	Cell culture	Serum	Cell culture	Cell culture	Cell culture	Serum
cell type	NA	SKBR3	NA	MCF7	MCF10A	MDAMB468	NA
medium	NA	EV-depleted serum	NA	EV-depleted serum	EV-depleted serum	EV-depleted serum	NA
condition	Control condition	Control condition	Breast cancer	Control condition	Control condition	Control condition	Control condition
separation protocol	(d)(U)C	(d)(U)C Filtration	(d)(U)C Filtration	(d)(U)C Filtration	(d)(U)C Filtration	(d)(U)C Filtration	(d)(U)C Filtration
Exp. nr.	5	1	3	7	2	4	6
EV-METRIC %	77	55	55	44	33	33	33

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