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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190002	1/6	Homo sapiens	MIA PaCa-2	DG (d)(U)C Filtration	Sachiko Matsumura	2018	78%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small extracellular vesicles 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 Filtration Protein markers EV: TSG101/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin non-EV: Histon H2B Proteomics no EV density (g/ml) 1.05-1.1 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 MIA PaCa-2 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes < 1.06 g/ml Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ MFGE8/ beta-actin/ HSP70/ CD81 Not detected EV-associated proteins TSG101/ Alix Not detected contaminants Histon H2B Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 136 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field Report size (nm) height: 39.1, diameter: 135.9

	EV190002	2/6	Homo sapiens	MIA PaCa-2	DG (d)(U)C Filtration	Sachiko Matsumura	2018	78%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small extracellular vesicles 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 Filtration Protein markers EV: TSG101/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin non-EV: Histon H2B Proteomics no EV density (g/ml) 1.05-1.1 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 MIA PaCa-2 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes ca. 1.1 g/ml Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ MFGE8/ beta-actin/ TSG101/ HSP70/ CD81 Not detected contaminants Histon H2B Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 124 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field Report size (nm) height: 30.1, diameter: 96.4

	EV190002	3/6	Homo sapiens	MIA PaCa-2	DG (d)(U)C Filtration	Sachiko Matsumura	2018	78%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small extracellular vesicles 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 Filtration Protein markers EV: TSG101/ Src/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin/ CD9 non-EV: Argonaute2/ Histon H2B Proteomics no EV density (g/ml) 1.05-1.1 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 MIA PaCa-2 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes < 1.06 g/ml Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ HSP70/ MFGE8/ Src/ beta-actin/ CD81 Not detected EV-associated proteins TSG101/ Alix Not detected contaminants Histon H2B/ Argonaute2 Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field Report size (nm) height: 50.5, length: 190.0

	EV190002	4/6	Homo sapiens	MIA PaCa-2	DG (d)(U)C Filtration	Sachiko Matsumura	2018	78%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small extracellular vesicles 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 Filtration Protein markers EV: TSG101/ Src/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin/ CD9 non-EV: Argonaute2/ Histon H2B Proteomics no EV density (g/ml) 1.05-1.1 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 MIA PaCa-2 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes ca. 1.1 g/ml Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ MFGE8/ Src/ beta-actin/ CD9/ CD63/ TSG101/ HSP70/ CD81 Not detected contaminants Histon H2B/ Argonaute2 Detected EV-associated proteins CD9/ CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 127 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field Report size (nm) height 32.1, length: 120.0

	EV190002	5/6	Homo sapiens	HT-29	DG (d)(U)C Filtration	Sachiko Matsumura	2018	67%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (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 Other / small extracellular vesicles 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 Filtration Protein markers EV: CD81/ EpCAM/ CD63/ CD9 non-EV: Proteomics no EV density (g/ml) 1.05-1.1 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 HT-29 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes < 1.06 g/ml Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins EpCAM/ CD9 Not detected EV-associated proteins CD81/ CD63 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 162 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field

	EV190002	6/6	Homo sapiens	HT-29	DG (d)(U)C Filtration	Sachiko Matsumura	2018	67%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (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 Other / small extracellular vesicles 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 Filtration Protein markers EV: CD81/ EpCAM/ CD63/ CD9 non-EV: Proteomics no EV density (g/ml) 1.05-1.1 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 HT-29 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes ca. 1.1 g/ml Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ EpCAM/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 142 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field

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EV-TRACK ID	EV190002
species	Homo sapiens
sample type	Cell culture
cell type	MIA PaCa-2	MIA PaCa-2	MIA PaCa-2	MIA PaCa-2	HT-29	HT-29
condition	Control condition	Control condition	Control condition	Control condition	Control condition	Control condition
separation protocol	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration	DG (d)(U)C Filtration
EV subtype	< 1.06 g/ml	ca. 1.1 g/ml	< 1.06 g/ml	ca. 1.1 g/ml	< 1.06 g/ml	ca. 1.1 g/ml
Exp. nr.	1	2	3	4	5	6
EV-METRIC %	78	78	78	78	67	67