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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV200036	5/16	Homo sapiens	Cell culture supernatant	DG (d)(U)C qEV	Juan Antonio Fafián-Labora	2020	56%
Study summary Full title Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage All authors Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen Journal Cell metab Abstract Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide) EV-METRIC 56% (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 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 Cell Name human skin primary fibroblasts Sample origin Progeria patients Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C qEV Protein markers EV: Alix/ GSTM2 non-EV: None Proteomics no EV density (g/ml) 1.074-1.106 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Progeria patients EV-producing cells human skin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type T-865 Speed (g) 100000 Duration (min) 720 Fraction volume (mL) 0.7 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 15 Pelleting-wash: duration (min) 80 Pelleting-wash: speed (g) T-865 Commercial kit qEV EV-subtype Distinction between multiple subtypes Size Used subtypes <200 nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ GSTM2 Characterization: Particle analysis NTA Report type Mean Reported size (nm) <200 EV concentration Yes Particle yield Number of particles of starting sample E08-E09

	EV200036	7/16	Homo sapiens	Cell culture supernatant	DG (d)(U)C qEV	Juan Antonio Fafián-Labora	2020	56%
Study summary Full title Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage All authors Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen Journal Cell metab Abstract Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide) EV-METRIC 56% (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 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 Cell Name primary human foreskin fibroblasts 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C qEV Protein markers EV: Alix/ GSTM2 non-EV: None Proteomics no EV density (g/ml) 1.074-1.106 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type T-865 Speed (g) 100000 Duration (min) 720 Fraction volume (mL) 0.7 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 15 Pelleting-wash: duration (min) 80 Pelleting-wash: speed (g) T-865 Commercial kit qEV EV-subtype Distinction between multiple subtypes Size Used subtypes <200 nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ GSTM2 Characterization: Particle analysis NTA Report type Mean Reported size (nm) <200 EV concentration Yes Particle yield Number of particles of starting sample E08-E09

	EV200036	9/16	Homo sapiens	Cell culture supernatant	DG (d)(U)C qEV	Juan Antonio Fafián-Labora	2020	56%
Study summary Full title Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage All authors Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen Journal Cell metab Abstract Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide) EV-METRIC 56% (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 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 Cell Name primary human foreskin fibroblasts Sample origin iRas Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C qEV Protein markers EV: Alix/ GSTM2 non-EV: None Proteomics no EV density (g/ml) 1.074-1.106 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition iRas EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type T-865 Speed (g) 100000 Duration (min) 720 Fraction volume (mL) 0.7 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 15 Pelleting-wash: duration (min) 80 Pelleting-wash: speed (g) T-865 Commercial kit qEV EV-subtype Distinction between multiple subtypes Size Used subtypes <200 nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ GSTM2 Characterization: Particle analysis NTA Report type Mean Reported size (nm) <200 EV concentration Yes Particle yield Number of particles of starting sample E08-E09

	EV200036	11/16	Homo sapiens	Cell culture supernatant	DG (d)(U)C qEV	Juan Antonio Fafián-Labora	2020	56%
Study summary Full title Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage All authors Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen Journal Cell metab Abstract Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide) EV-METRIC 56% (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 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 Cell Name primary human foreskin fibroblasts Sample origin iRas+GSTM2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C qEV Protein markers EV: Alix/ GSTM2 non-EV: None Proteomics no EV density (g/ml) 1.074-1.106 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition iRas+GSTM2 EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type T-865 Speed (g) 100000 Duration (min) 720 Fraction volume (mL) 0.7 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 15 Pelleting-wash: duration (min) 80 Pelleting-wash: speed (g) T-865 Commercial kit qEV EV-subtype Distinction between multiple subtypes Size Used subtypes <200 nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ GSTM2 Characterization: Particle analysis NTA Report type Mean Reported size (nm) <200 EV concentration Yes Particle yield Number of particles of starting sample E08-E09

	EV200036	13/16	Homo sapiens	Cell culture supernatant	DG (d)(U)C qEV	Juan Antonio Fafián-Labora	2020	56%
Study summary Full title Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage All authors Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen Journal Cell metab Abstract Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide) EV-METRIC 56% (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 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 Cell Name primary human foreskin fibroblasts Sample origin iC+GSTM2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C qEV Protein markers EV: Alix/ GSTM2 non-EV: None Proteomics no EV density (g/ml) 1.074-1.106 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition iC+GSTM2 EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type T-865 Speed (g) 100000 Duration (min) 720 Fraction volume (mL) 0.7 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 15 Pelleting-wash: duration (min) 80 Pelleting-wash: speed (g) T-865 Commercial kit qEV EV-subtype Distinction between multiple subtypes Size Used subtypes <200 nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ GSTM2 Characterization: Particle analysis NTA Report type Mean Reported size (nm) <200 EV concentration Yes Particle yield Number of particles of starting sample E08-E09

	EV200036	15/16	Homo sapiens	Cell culture supernatant	DG (d)(U)C qEV	Juan Antonio Fafián-Labora	2020	56%
Study summary Full title Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage All authors Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen Journal Cell metab Abstract Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide) EV-METRIC 56% (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 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 Cell Name primary human foreskin fibroblasts Sample origin mCherry-CD63 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C qEV Protein markers EV: Alix/ GSTM2 non-EV: None Proteomics no EV density (g/ml) 1.074-1.106 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition mCherry-CD63 EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 15 Wash: time (min) 80 Wash: Rotor Type T-865 Wash: speed (g) 100000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 60% Total gradient volume, incl. sample (mL) 5.5 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type T-865 Speed (g) 100000 Duration (min) 720 Fraction volume (mL) 0.7 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 80 Pelleting: rotor type T-865 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 15 Pelleting-wash: duration (min) 80 Pelleting-wash: speed (g) T-865 Commercial kit qEV EV-subtype Distinction between multiple subtypes Size Used subtypes <200 nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ GSTM2 Characterization: Particle analysis NTA Report type Mean Reported size (nm) <200 EV concentration Yes Particle yield Number of particles of starting sample E08-E09

	EV200034	1/1	Homo sapiens	Cell culture supernatant	(d)(U)C DG Filtration	Xiaohui Chen	2020	56%
Study summary Full title High-Fidelity Determination and Tracing of Small Extracellular Vesicle Cargoes All authors Xiaohui Chen, Mei Jia, Lianhua Liu, Xiaopei Qiu, Hong Zhang, Xingle Yu, Wei Gu, Guangchao Qing, Qingmei Li, Xiaolin Hu, Ruixuan Wang, Xianxian Zhao, Liangliang Zhang, Xianfeng Wang, Colm Durkan, Nan Wang, Guixue Wang, Yang Luo Journal Small Abstract Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for eluci (show more...)Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high-fidelity determination of sEVs' molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular-Guided Hairpin-Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single-nucleotide discrimination as well as wash-free fluorescence readout using a hairpin probe, the proposed approach enables high-fidelity fluorescence visualization of sEVs' microRNA without RNA extraction or leakage, demonstrating the potential of on-site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV-derived fundamental biological studies and molecular diagnostics. (hide) EV-METRIC 56% (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 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 Cell Name A549 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: TSG101/ CD9/ CD81/ CD63/ actin-beta non-EV: None Proteomics no EV density (g/ml) 1.15 Show all info Study aim New methodological development/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells A549 EV-harvesting Medium Serum free medium Cell viability Yes Cell viability (%) Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Bottom-up Rotor type Not specified Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Ultracentrifugation Pelleting: duration (min) 60 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins TSG101/ CD9/ CD81/ CD63/ actin-beta Flow cytometry Hardware adjustments Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Modus Reported size (nm) 104 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200024	1/2	Homo sapiens	Blood plasma	(d)(U)C	Otahal, Alexander	2020	56%
Study summary Full title Characterization and Chondroprotective Effects of Extracellular Vesicles From Plasma- and Serum-Based Autologous Blood-Derived Products for Osteoarthritis Therapy All authors Alexander Otahal, Karina Kramer, Olga Kuten-Pella, René Weiss, Christoph Stotter, Zsombor Lacza, Viktoria Weber, Stefan Nehrer and Andrea De Luna Journal Front. Bioeng. Biotechnol. Abstract Autologous blood products gain increasing interest in the field of regenerative medicine as well as (show more...)Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA. (hide) EV-METRIC 56% (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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD63/ CD9 non-EV: APOA1/ APOB100 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-80 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD9/ CD63/ Alix Not detected contaminants APOA1/ APOB100 Characterization: Particle analysis NTA Report type Modus Reported size (nm) 160 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Gallios Hardware adjustment Calibration bead size 1

	EV200024	2/2	Homo sapiens	Serum	(d)(U)C	Otahal, Alexander	2020	56%
Study summary Full title Characterization and Chondroprotective Effects of Extracellular Vesicles From Plasma- and Serum-Based Autologous Blood-Derived Products for Osteoarthritis Therapy All authors Alexander Otahal, Karina Kramer, Olga Kuten-Pella, René Weiss, Christoph Stotter, Zsombor Lacza, Viktoria Weber, Stefan Nehrer and Andrea De Luna Journal Front. Bioeng. Biotechnol. Abstract Autologous blood products gain increasing interest in the field of regenerative medicine as well as (show more...)Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA. (hide) EV-METRIC 56% (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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD63/ CD9 non-EV: APOA1/ APOB100 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-80 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD9/ CD63/ Alix Not detected contaminants APOA1/ APOB100 Characterization: Particle analysis NTA Report type Modus Reported size (nm) 170 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Gallios Hardware adjustment Calibration bead size 1 Report type Modus Reported size (nm) 170

	EV200016	1/2	Bos taurus	Cell culture supernatant	(d)(U)C ExoQuick Filtration	Samuel Gebremedhn	2020	56%
Study summary Full title Extracellular vesicles shuttle protective messages against heat stress in bovine granulosa cells All authors Samuel Gebremedhn, Ahmed Gad, Hoda Samir Aglan, Jozef Laurincik, Radek Prochazka, Dessie Salilew-Wondim, Michael Hoelker, Karl Schellander, Dawit Tesfaye Journal Sci Rep Abstract Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, wh (show more...)Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS. (hide) EV-METRIC 56% (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 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 Cell Name Ovarian granulosa cells Sample origin Granulosa cells subjected to normal temperature (37OC) Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick Filtration Protein markers EV: CD63 non-EV: Cytochrome C Proteomics no Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type Cell culture supernatant Sample Condition Granulosa cells subjected to normal temperature (37OC) EV-producing cells Ovarian granulosa cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability Yes Cell viability (%) Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 5 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis PMID previous EV protein analysis Extra characterization Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Not detected contaminants Cytochrome C Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Modus Reported size (nm) 131 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5.98E+08 EM EM-type Transmission-EM Image type Close-up

	EV200016	2/2	Bos taurus	Cell culture supernatant	(d)(U)C ExoQuick Filtration	Samuel Gebremedhn	2020	56%
Study summary Full title Extracellular vesicles shuttle protective messages against heat stress in bovine granulosa cells All authors Samuel Gebremedhn, Ahmed Gad, Hoda Samir Aglan, Jozef Laurincik, Radek Prochazka, Dessie Salilew-Wondim, Michael Hoelker, Karl Schellander, Dawit Tesfaye Journal Sci Rep Abstract Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, wh (show more...)Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS. (hide) EV-METRIC 56% (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 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 Cell Name Ovarian granulosa cells Sample origin Granulosa cells subjected to higher temperature (42OC) Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick Filtration Protein markers EV: CD63 non-EV: Cytochrome Proteomics no Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type Cell culture supernatant Sample Condition Granulosa cells subjected to higher temperature (42OC) EV-producing cells Ovarian granulosa cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability Yes Cell viability (%) Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 70 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 5 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis PMID previous EV protein analysis Extra characterization Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Not detected contaminants Cytochrome Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Modus Reported size (nm) 129 EV concentration Yes Particle yield Yes, as number of particles per million cells 7.23E+08 EM EM-type Transmission-EM Image type Close-up

	EV200012	2/2	Rattus norvegicus	Cell culture supernatant	UF	Doreen Matthies	2020	56%
Study summary Full title Microdomains form on the luminal face of neuronal extracellular vesicle membranes All authors Doreen Matthies, Nathanael Y J Lee, Ian Gatera, H Amalia Pasolli, Xiaowei Zhao, Hui Liu, Deepika Walpita, Zhe Liu, Zhiheng Yu, Maria S Ioannou Journal Sci Rep Abstract Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been imp (show more...)Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology. (hide) EV-METRIC 56% (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 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 Cell Name Primary cortical neurons 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration Protein markers EV: tubulin/ Flotillin1/ syntenin non-EV: gp96 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Rattus norvegicus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Primary cortical neurons EV-harvesting Medium Serum free medium Cell number specification No Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis PMID previous EV protein analysis Extra characterization Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ syntenin Not detected EV-associated proteins tubulin Not detected contaminants gp96 PMID previous EV particle analysis Extra particle analysis EM EM-type Cryo-EM Image type Close-up Report size (nm) 147.82+/-95.72 EV concentration Yes

	EV200000	1/4	Homo sapiens	pleural effusion	(d)(U)C	Ping, Luo	2020	56%
Study summary Full title Metabolic characteristics of large and small extracellular vesicles from pleural effusion reveal biomarker candidates for the diagnosis of tuberculosis and malignancy All authors Ping Luo, Kaimin Mao, Juanjuan Xu, Feng Wu, Xuan Wang, Sufei Wang, Mei Zhou, Limin Duan, Qi Tan, Guangzhou Ma, Guanghai Yang, Ronghui Du, Hai Huang, Qi Huang, Yumei Li, Mengfei Guo, Yang Jin Journal J Extracell Vesicles Abstract Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of (show more...)Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of tuberculosis pleural effusion (TPE) and malignancy pleural effusion (MPE) remain challenging. Although extracellular vesicles (EVs) have been confirmed as promising sources of disease biomarkers, little is known about the metabolite compositions of its subpopulations and their roles in the diagnosis of pleural effusion. Here, we performed metabolomics and lipidomics analysis to investigate the metabolite characteristics of two EV subpopulations derived from pleural effusion by differential ultracentrifugation, namely large EVs (lEVs, pelleted at 20,000 × g) and small EVs (sEVs, pelleted at 110,000 × g), and assessed their metabolite differences between tuberculosis and malignancy. A total of 579 metabolites, including amino acids, acylcarnitines, organic acids, steroids, amides and various lipid species, were detected. The results showed that the metabolic profiles of lEVs and sEVs overlapped with and difference from each other but significantly differed from those of pleural effusion. Additionally, different type of vesicles and pleural effusion showed unique metabolic enrichments. Furthermore, lEVs displayed more significant and larger metabolic alterations between the tuberculosis and malignancy groups, and their differential metabolites were more closely related to clinical parameters than those of sEV. Finally, a panel of four biomarker candidates, including phenylalanine, leucine, phosphatidylcholine 35:0, and sphingomyelin 44:3, in pleural lEVs was defined based on the comprehensive discovery and validation workflow. This panel showed high performance for distinguishing TPE and MPE, particularly in patients with delayed or missed diagnosis, such as the area under the receiver-operating characteristic curve (AUC) >0.95 in both sets. We conducted comprehensive metabolic profiling analysis of EVs, and further explored the metabolic reprogramming of tuberculosis and malignancy at the level of metabolites in lEVs and sEVs, providing insight into the mechanism of pleural effusion, and identifying novel biomarkers for diagnosing TPE and MPE. (hide) EV-METRIC 56% (57th 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 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 pleural effusion Sample origin tuberculosis Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ CD9 non-EV: calnexin/ GM130 Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type pleural effusion Sample Condition tuberculosis Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 EV-subtype Distinction between multiple subtypes Size Used subtypes 50-200 nm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ TSG101/ CD81 Not detected EV-associated proteins CD9 Not detected contaminants calnexin/ GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 136.1 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 182567942

	EV200000	2/4	Homo sapiens	pleural effusion	(d)(U)C	Ping, Luo	2020	56%
Study summary Full title Metabolic characteristics of large and small extracellular vesicles from pleural effusion reveal biomarker candidates for the diagnosis of tuberculosis and malignancy All authors Ping Luo, Kaimin Mao, Juanjuan Xu, Feng Wu, Xuan Wang, Sufei Wang, Mei Zhou, Limin Duan, Qi Tan, Guangzhou Ma, Guanghai Yang, Ronghui Du, Hai Huang, Qi Huang, Yumei Li, Mengfei Guo, Yang Jin Journal J Extracell Vesicles Abstract Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of (show more...)Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of tuberculosis pleural effusion (TPE) and malignancy pleural effusion (MPE) remain challenging. Although extracellular vesicles (EVs) have been confirmed as promising sources of disease biomarkers, little is known about the metabolite compositions of its subpopulations and their roles in the diagnosis of pleural effusion. Here, we performed metabolomics and lipidomics analysis to investigate the metabolite characteristics of two EV subpopulations derived from pleural effusion by differential ultracentrifugation, namely large EVs (lEVs, pelleted at 20,000 × g) and small EVs (sEVs, pelleted at 110,000 × g), and assessed their metabolite differences between tuberculosis and malignancy. A total of 579 metabolites, including amino acids, acylcarnitines, organic acids, steroids, amides and various lipid species, were detected. The results showed that the metabolic profiles of lEVs and sEVs overlapped with and difference from each other but significantly differed from those of pleural effusion. Additionally, different type of vesicles and pleural effusion showed unique metabolic enrichments. Furthermore, lEVs displayed more significant and larger metabolic alterations between the tuberculosis and malignancy groups, and their differential metabolites were more closely related to clinical parameters than those of sEV. Finally, a panel of four biomarker candidates, including phenylalanine, leucine, phosphatidylcholine 35:0, and sphingomyelin 44:3, in pleural lEVs was defined based on the comprehensive discovery and validation workflow. This panel showed high performance for distinguishing TPE and MPE, particularly in patients with delayed or missed diagnosis, such as the area under the receiver-operating characteristic curve (AUC) >0.95 in both sets. We conducted comprehensive metabolic profiling analysis of EVs, and further explored the metabolic reprogramming of tuberculosis and malignancy at the level of metabolites in lEVs and sEVs, providing insight into the mechanism of pleural effusion, and identifying novel biomarkers for diagnosing TPE and MPE. (hide) EV-METRIC 56% (57th 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 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 pleural effusion Sample origin tuberculosis Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ TSG101/ CD63/ CD9 non-EV: calnexin/ GM130 Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type pleural effusion Sample Condition tuberculosis Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 EV-subtype Distinction between multiple subtypes Size Used subtypes 200-1000 nm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected EV-associated proteins CD81 Not detected contaminants calnexin/ GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 224.2 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 244682871

	EV190089	1/1	Homo sapiens	Cell culture supernatant	(d)(U)C	Guowen Hu	2020	56%
Study summary Full title ESC‐sEVs Rejuvenate Senescent Hippocampal NSCs by Activating Lysosomes to Improve Cognitive Dysfunction in Vascular Dementia All authors Guowen Hu, Yuguo Xia, Juntao Zhang, Yu Chen, Ji Yuan, Xin Niu, Bizeng Zhao, Qing Li, Yang Wang, Zhifeng Deng Journal Advanced Science Abstract Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism (show more...)Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism is unclear and there is still lack of effective medications. In this study, the VD rats exhibit a progressive cognitive impairment, as well as a time‐related increasing in hippocampal neural stem cells (H‐NSCs) senescence, lost and neurogenesis decline. Then, embryonic stem cell‐derived small extracellular vesicles (ESC‐sEVs) are intravenously injected into VD rats. ESC‐sEVs treatment significantly alleviates H‐NSCs senescence, recovers compromised proliferation and neuron differentiation capacity, and reverses cognitive impairment. By microarray analysis and RT‐qPCR it is identified that several miRNAs including miR‐17‐5p, miR‐18a‐5p, miR‐21‐5p, miR‐29a‐3p, and let‐7a‐5p, that can inhibit mTORC1 activation, exist in ESC‐sEVs. ESC‐sEVs rejuvenate H‐NSCs senescence partly by transferring these miRNAs to inhibit mTORC1 activation, promote transcription factor EB (TFEB) nuclear translocation and lysosome resumption. Taken together, these data indicate that H‐NSCs senescence cause cell depletion, neurogenesis reduction, and cognitive impairment in VD. ESC‐sEVs treatment ameliorates H‐NSCs senescence by inhibiting mTORC1 activation, and promoting TFEB nuclear translocation and lysosome resumption, thereby reversing senescence‐related neurogenesis dysfunction and cognitive impairment in VD. The application of ESC‐sEVs may be a novel cell‐free therapeutic tool for patients with VD, as well as other aging‐related diseases. (hide) EV-METRIC 56% (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 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 Cell Name Embryonic stem cells 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ CD9 non-EV: GM130 Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Embryonic stem cells EV-harvesting Medium Serum free medium Separation Method Differential ultracentrifugation centrifugation steps Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 114 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38.5 Wash: time (min) 114 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected contaminants GM130

	EV190069	3/4	Homo sapiens	Cell culture supernatant	DG (d)(U)C	Mariscal, Javier	2020	56%
Study summary Full title Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles All authors Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide) EV-METRIC 56% (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 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 Cell Name DU145 Sample origin DIAPH3 Knock down Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD9/ HSPA5 non-EV: Proteomics no EV density (g/ml) 1.1 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition DIAPH3 Knock down EV-producing cells DU145 EV-harvesting Medium Serum free medium Cell viability Yes Cell viability (%) Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Obtain an EV pellet : Yes Pelleting: time(min) 30 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 30% Total gradient volume, incl. sample (mL) 16.2 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 28 Speed (g) 100000 Duration (min) 230 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Other;Pierce 660 nm Western Blot Detected EV-associated proteins HSPA5 Not detected EV-associated proteins CD9 Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 1650 EV concentration Yes

	EV190069	4/4	Homo sapiens	Cell culture supernatant	DG (d)(U)C	Mariscal, Javier	2020	56%
Study summary Full title Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles All authors Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide) EV-METRIC 56% (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 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 Cell Name DU145 Sample origin DIAPH3 Knock down Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD9/ HSPA5 non-EV: Proteomics no EV density (g/ml) 1.1 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition DIAPH3 Knock down EV-producing cells DU145 EV-harvesting Medium Serum free medium Cell viability Yes Cell viability (%) Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 30% Total gradient volume, incl. sample (mL) 16.2 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 28 Speed (g) 100000 Duration (min) 230 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Other;Pierce 660 nm Western Blot Detected EV-associated proteins CD9 Not detected EV-associated proteins HSPA5 Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 116 EV concentration Yes

	EV190066	1/2	Homo sapiens	Cell culture supernatant	(d)(U)C qEV	Gori, Alessandro	2020	56%
Study summary Full title Membrane-binding peptides for extracellular vesicles on-chip analysis All authors Alessandro Gori, Alessandro Romanato, Bergamaschi Greta, Alessandro Strada, Paola Gagni, Roberto Frigerio, Dario Brambilla, Riccardo Vago, Silvia Galbiati, Silvia Picciolini, Marzia Bedoni, George G. Daaboul, Marcella Chiari, and Marina Creticha Journal J Extracell Vesicles Abstract Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extrac (show more...)Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis. (hide) EV-METRIC 56% (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 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 Cell Name HEK 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV Protein markers EV: TSG101/ Alix/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells HEK EV-harvesting Medium Not specified Separation Method Differential ultracentrifugation centrifugation steps Equal to or above 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Surespin 630 (17 ml) Pelleting: speed (g) 28400 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Characterization: Particle analysis NTA Report type Mean Reported size (nm) 203 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV190066	2/2	Homo sapiens	Serum	(d)(U)C qEV	Gori, Alessandro	2020	56%
Study summary Full title Membrane-binding peptides for extracellular vesicles on-chip analysis All authors Alessandro Gori, Alessandro Romanato, Bergamaschi Greta, Alessandro Strada, Paola Gagni, Roberto Frigerio, Dario Brambilla, Riccardo Vago, Silvia Galbiati, Silvia Picciolini, Marzia Bedoni, George G. Daaboul, Marcella Chiari, and Marina Creticha Journal J Extracell Vesicles Abstract Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extrac (show more...)Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis. (hide) EV-METRIC 56% (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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV Protein markers EV: TSG101/ Alix/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Equal to or above 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 41900 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101 Not detected EV-associated proteins TSG101/ CD63/ CD9/ Alix Characterization: Particle analysis NTA Report type Mean Reported size (nm) 208 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV190064	2/10	Homo sapiens	Urine	(d)(U)C Filtration	Dhondt B	2020	56%
Study summary Full title Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. All authors Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A. Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide) EV-METRIC 56% (90th 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 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 Urine 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: Alix/ Flotillin1/ CD9 non-EV: Tamm-Horsfall protein Proteomics no Show all info Study aim Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Sample Condition Control condition Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 16 Wash: time (min) 70 Wash: Rotor Type SW 32.1 Ti Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD9 Detected contaminants Tamm-Horsfall protein Characterization: Particle analysis NTA Report type Mean Reported size (nm) 226.2 EV concentration Yes

	EV190060	1/4	Homo sapiens	Blood plasma	(d)(U)C	Mari Palviainen	2020	56%
Study summary Full title Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery All authors Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander Journal PLoS One Abstract Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide) EV-METRIC 56% (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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD61/ CD41/ phosphatidylserine/ CD235a/ CD9 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD9/ CD41/ TSG101 Flow cytometry aspecific beads Detected EV-associated proteins CD61/ CD235a/ phosphatidylserine Proteomics Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Particle analysis NTA Report type Modus Reported size (nm) 107-145 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Apogee A50 Hardware adjustment calibration done with apogee Mix beads 80-1300 nm Calibration bead size 80 EM EM-type Transmission-EM Image type Close-up

	EV190060	2/4	Homo sapiens	Blood plasma	(d)(U)C	Mari Palviainen	2020	56%
Study summary Full title Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery All authors Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander Journal PLoS One Abstract Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide) EV-METRIC 56% (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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD61/ CD41/ phosphatidylserine/ CD235a/ CD9 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD9/ CD41/ TSG101 Flow cytometry aspecific beads Detected EV-associated proteins CD61/ CD235a/ phosphatidylserine Proteomics Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Particle analysis NTA Report type Modus Reported size (nm) 117-162 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type apogee A50 Hardware adjustment calibration done with apogee Mix beads 80-1300 nm Calibration bead size 80 Report type Not Reported EM EM-type Transmission-EM Image type Close-up

	EV190060	3/4	Homo sapiens	Blood plasma	(d)(U)C	Mari Palviainen	2020	56%
Study summary Full title Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery All authors Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander Journal PLoS One Abstract Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide) EV-METRIC 56% (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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD61/ CD41/ phosphatidylserine/ Cd235a/ CD9 non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD9/ TSG101/ CD41 Flow cytometry aspecific beads Detected EV-associated proteins CD61/ Cd235a/ phosphatidylserine Proteomics Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Particle analysis NTA Report type Modus Reported size (nm) 106-160 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Apogee A50 Hardware adjustment calibration done with apogee Mix beads 80-1300 nm Calibration bead size 80 Report type Not Reported EM EM-type Transmission-EM Image type Close-up

	EV190060	4/4	Homo sapiens	Serum	(d)(U)C	Mari Palviainen	2020	56%
Study summary Full title Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery All authors Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander Journal PLoS One Abstract Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide) EV-METRIC 56% (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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD235a/ CD41/ CD9/ phosphatidylserine non-EV: Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 90 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 20 Wash: time (min) 90 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins CD41/ CD9/ TSG101 Flow cytometry aspecific beads Detected EV-associated proteins CD41/ CD235a/ phosphatidylserine Proteomics Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Particle analysis NTA Report type Modus Reported size (nm) 88-128 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Apogee A50 Hardware adjustment calibration done with apogee Mix beads 80-1300 nm Calibration bead size 80 Report type Not Reported EM EM-type Transmission-EM Image type Close-up

	EV190059	1/1	Homo sapiens	Cell culture supernatant	(d)(U)C Filtration	Gong, Liangzhi	2020	56%
Study summary Full title Human ESC-sEVs alleviate age-related bone loss by rejuvenating senescent bone marrow-derived mesenchymal stem cells All authors Liangzhi Gong, Bi Chen, Juntao Zhang, Yongjin Sun, Ji Yuan, Xin Niu, Guowen Hu, Yu Chen, Zongping Xie, Zhifeng Deng, Qing Li, Yang Wang Journal J Extracell Vesicles Abstract Tissue-resident stem cell senescence leads to stem cell exhaustion, which is a major cause of physio (show more...)Tissue-resident stem cell senescence leads to stem cell exhaustion, which is a major cause of physiological and pathological ageing. Stem cell-derived extracellular vesicles (SC-EVs) have been reported in preclinical studies to possess therapeutic potential for diverse diseases. However, whether SC-EVs can rejuvenate senescent tissue stem cells to prevent age-related disorders still remains unknown. Here, we show that chronic application of human embryonic stem cell-derived small extracellular vesicles (hESC-sEVs) rescues the function of senescent bone marrow mesenchymal stem cells (BM-MSCs) and prevents age-related bone loss in ageing mice. Transcriptome analysis revealed that hESC-sEVs treatment upregulated the expression of genes involved in antiaging, stem cell proliferation and osteogenic differentiation in BM-MSCs. Furthermore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified 4122 proteins encapsulated in hESC-sEVs. Bioinformatics analysis predicted that the protein components in the hESCs-sEVs function in a synergistic way to induce the activation of several canonical signalling pathways, including Wnt, Sirtuin, AMPK, PTEN signalling, which results in the upregulation of antiaging genes in BM-MSCs and then the recovery of senescent BM-MSCs function. Collectively, our findings reveal the effect of hESC-sEVs in reversing BM-MSCs senescence and age-related osteogenic dysfunction, thereby preventing age-related bone loss. Because hESC-sEVs could alleviate senescence of tissue-resident stem cells, they might be promising therapeutic candidates for age-related diseases. (hide) EV-METRIC 56% (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 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 Cell Name H9 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ Alix/ CD63/ CD9 non-EV: GM130 Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells H9 EV-harvesting Medium Serum free medium Cell viability Yes Cell viability (%) Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 114 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 114 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm0.2µm > x > 0.1µm EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Not detected contaminants GM130 Proteomics Proteomics database No Other particle analysis name(1) qNano Report type Size range/distribution Report size 75-200 EV-concentration Yes

	EV200117	2/6	Homo sapiens	Cell culture supernatant	(d)(U)C UF qEV	Cocozza, Federico	2020	50%
Study summary Full title Extracellular vesicles containing ACE2 efficiently prevent infection by SARS‐CoV‐2 Spike protein‐containing virus All authors Federico Cocozza, Nathalie Névo, Ester Piovesana, Xavier Lahaye, Julian Buchrieser, Olivier Schwartz, Nicolas Manel, Mercedes Tkach, Clotilde Théry, Lorena Martin‐Jaular Journal J Extracell Vesicles Abstract SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 an (show more...)SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2‐EVs are efficient decoys for SARS‐CoV‐2 S protein‐containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2. (hide) EV-METRIC 50% (80th 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 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 Cell Name HEK Sample origin HEK MOCK Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: CD81/ ACE2/ ADAM10/ CD63/ syntenin-1/ HSP70 non-EV: AChe Proteomics no Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition HEK MOCK EV-producing cells HEK EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability Yes Cell viability (%) Yes Cell number specification Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ syntenin-1/ ACE2/ ADAM10/ CD81/ HSP70 Not detected contaminants AChe ELISA Detected EV-associated proteins ACE2 Flow cytometry Hardware adjustments Detected EV-associated proteins CD63/ CD81/ syntenin-1 Detected EV-associated proteins CD81/ CD63/ syntenin-1 Characterization: Particle analysis NTA Report type Median Reported size (nm) 141.8 EV concentration Yes Particle yield Yes, as number of particles per million cells 5.00E+08

	EV200117	3/6	Homo sapiens	Cell culture supernatant	(d)(U)C UF qEV	Cocozza, Federico	2020	50%
Study summary Full title Extracellular vesicles containing ACE2 efficiently prevent infection by SARS‐CoV‐2 Spike protein‐containing virus All authors Federico Cocozza, Nathalie Névo, Ester Piovesana, Xavier Lahaye, Julian Buchrieser, Olivier Schwartz, Nicolas Manel, Mercedes Tkach, Clotilde Théry, Lorena Martin‐Jaular Journal J Extracell Vesicles Abstract SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 an (show more...)SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2‐EVs are efficient decoys for SARS‐CoV‐2 S protein‐containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2. (hide) EV-METRIC 50% (80th 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 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 Cell Name HEK Sample origin HEK ACE2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: CD81/ ACE2/ ADAM10/ CD63/ syntenin-1/ HSP70 non-EV: AChe Proteomics no Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition HEK ACE2 EV-producing cells HEK EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability Yes Cell viability (%) Yes Cell number specification Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ syntenin-1/ ACE2/ ADAM10/ CD81/ HSP70 Not detected contaminants AChe ELISA Detected EV-associated proteins ACE2 Flow cytometry Hardware adjustments Detected EV-associated proteins CD63/ CD81/ syntenin-1 Detected EV-associated proteins CD81/ CD63/ syntenin-1 Characterization: Particle analysis NTA Report type Median Reported size (nm) 145.5 EV concentration Yes Particle yield Yes, as number of particles per million cells 7.00E+08

	EV200117	4/6	Homo sapiens	Cell culture supernatant	(d)(U)C UF qEV	Cocozza, Federico	2020	50%
Study summary Full title Extracellular vesicles containing ACE2 efficiently prevent infection by SARS‐CoV‐2 Spike protein‐containing virus All authors Federico Cocozza, Nathalie Névo, Ester Piovesana, Xavier Lahaye, Julian Buchrieser, Olivier Schwartz, Nicolas Manel, Mercedes Tkach, Clotilde Théry, Lorena Martin‐Jaular Journal J Extracell Vesicles Abstract SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 an (show more...)SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2‐EVs are efficient decoys for SARS‐CoV‐2 S protein‐containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2. (hide) EV-METRIC 50% (80th 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 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 Cell Name HEK Sample origin HEK ACE2 and TMPRSS2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: CD81/ ACE2/ ADAM10/ CD63/ syntenin-1/ HSP70 non-EV: AChe Proteomics no Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition HEK ACE2 and TMPRSS2 EV-producing cells HEK EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability Yes Cell viability (%) Yes Cell number specification Yes Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins syntenin-1/ ACE2/ ADAM10/ CD63/ CD81/ HSP70 Not detected contaminants AChe ELISA Detected EV-associated proteins ACE2 Flow cytometry Hardware adjustments Detected EV-associated proteins CD63/ CD81/ syntenin-1 Detected EV-associated proteins CD81/ CD63/ syntenin-1 Characterization: Particle analysis NTA Report type Median Reported size (nm) 153.9 EV concentration Yes Particle yield Yes, as number of particles per million cells 6.00E+08

	EV200068	1/5	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Linda Hofmann	2020	50%
Study summary Full title The Potential of CD16 on Plasma-Derived Exosomes as a Liquid Biomarker in Head and Neck Cancer All authors Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki Journal Int J Mol Sci Abstract Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide) EV-METRIC 50% (85th 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 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 exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C SEC (non-commercial) Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9/ CD16 non-EV: Grp94/ ApoA1 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected contaminants ApoA1/ Grp94 Flow cytometry aspecific beads Detected EV-associated proteins CD16 Flow cytometry specific beads Selected surface protein(s) CD44v3 Detected EV-associated proteins CD16 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-150 EM EM-type Transmission-EM Image type Wide-field

	EV200068	2/5	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Linda Hofmann	2020	50%
Study summary Full title The Potential of CD16 on Plasma-Derived Exosomes as a Liquid Biomarker in Head and Neck Cancer All authors Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki Journal Int J Mol Sci Abstract Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide) EV-METRIC 50% (85th 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 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 HNSCC Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C SEC (non-commercial) Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9/ CD16 non-EV: Grp94/ ApoA1 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition HNSCC Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected contaminants ApoA1/ Grp94 Flow cytometry aspecific beads Detected EV-associated proteins CD16 Flow cytometry specific beads Selected surface protein(s) CD44v3 Detected EV-associated proteins CD16

	EV200063	1/2	Homo sapiens	Blood plasma	qEV	Silvia Picciolini	2020	50%
Study summary Full title An SPRi-based biosensor pilot study: Analysis of multiple circulating extracellular vesicles and hippocampal volume in Alzheimer’s disease All authors Silvia Picciolini,Alice Gualerzi,Cristiano Carlomagno,Monia Cabinio,Stefano Sorrentino,Francesca Baglio,Marzia Bedoni Journal ACS Nano Abstract One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible (show more...)One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible and sensitive biomarkers for the diagnosis, the prediction of the disease progression rate and the evaluation of rehabilitative and pharmacological treatments. Extracellular Vesicles (EVs) are nanoscales particles released by body cells studied as promising biomarkers of AD as they are involved in the onset and progression of the disease. In the strive for a reliable and sensitive method to analyze EVs, we applied our recently developed biosensor based on Surface Plasmon Resonance imaging (SPRi) technology for the identification and profiling of neural EVs populations circulating in the plasma of 10 AD patients and 10 healthy subjects. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia and oligodendrocytes, and to evaluate the presence and the relative amount of specific surface molecules related to pathological processes including translocator protein (TSPO), β-Amyloid and ganglioside M1. (hide) EV-METRIC 50% (85th 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 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture qEV Protein markers EV: CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB non-EV: Ig Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation Method Commercial kit qEV Characterization: Protein analysis Protein Concentration Method BCA Detected EV-associated proteins CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB Not detected contaminants Ig Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 166,45 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV200063	2/2	Homo sapiens	Blood plasma	qEV	Silvia Picciolini	2020	50%
Study summary Full title An SPRi-based biosensor pilot study: Analysis of multiple circulating extracellular vesicles and hippocampal volume in Alzheimer’s disease All authors Silvia Picciolini,Alice Gualerzi,Cristiano Carlomagno,Monia Cabinio,Stefano Sorrentino,Francesca Baglio,Marzia Bedoni Journal ACS Nano Abstract One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible (show more...)One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible and sensitive biomarkers for the diagnosis, the prediction of the disease progression rate and the evaluation of rehabilitative and pharmacological treatments. Extracellular Vesicles (EVs) are nanoscales particles released by body cells studied as promising biomarkers of AD as they are involved in the onset and progression of the disease. In the strive for a reliable and sensitive method to analyze EVs, we applied our recently developed biosensor based on Surface Plasmon Resonance imaging (SPRi) technology for the identification and profiling of neural EVs populations circulating in the plasma of 10 AD patients and 10 healthy subjects. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia and oligodendrocytes, and to evaluate the presence and the relative amount of specific surface molecules related to pathological processes including translocator protein (TSPO), β-Amyloid and ganglioside M1. (hide) EV-METRIC 50% (85th 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 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 Alzheimer disease Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture qEV Protein markers EV: CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB non-EV: Ig Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Alzheimer disease Separation Method Commercial kit qEV Characterization: Protein analysis Protein Concentration Method BCA Detected EV-associated proteins CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB Not detected contaminants Ig Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 183,28 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV190100	1/10	Homo sapiens	Cell culture supernatant	ExoQuick	Chaoliang Liao	2020	50%
Study summary Full title Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells All authors Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang Journal J Pharm Sci Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide) EV-METRIC 50% (80th 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 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 Cell Name CNE1 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Protein markers EV: HSP70/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells CNE1 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ HSP70 Not detected contaminants Calnexin Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 21.04-255.6 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 80-100

	EV190100	2/10	Homo sapiens	Cell culture supernatant	ExoQuick	Chaoliang Liao	2020	50%
Study summary Full title Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells All authors Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang Journal J Pharm Sci Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide) EV-METRIC 50% (80th 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 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 Cell Name CNE1-LMP1 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Protein markers EV: HSP70/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells CNE1-LMP1 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ HSP70 Not detected contaminants Calnexin EM EM-type Transmission-EM Image type Close-up Report size (nm) 80-100

	EV190100	6/10	Homo sapiens	Cell culture supernatant	ExoQuick	Chaoliang Liao	2020	50%
Study summary Full title Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells All authors Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang Journal J Pharm Sci Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide) EV-METRIC 50% (80th 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 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 Cell Name HKI 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Protein markers EV: HSP70/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells HKI EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ HSP70 Not detected contaminants Calnexin Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 24.3-199.2 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 80-100

	EV190100	8/10	Homo sapiens	Cell culture supernatant	ExoQuick	Chaoliang Liao	2020	50%
Study summary Full title Epstein-Barr virus-encoded latent membrane protein 1 promotes extracellular vesicle secretion through syndecan-2 and synaptotagmin-like-4 in nasopharyngeal carcinoma cells All authors Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang Journal J Pharm Sci Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide) EV-METRIC 50% (80th 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 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 Cell Name C666-1 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Protein markers EV: HSP70/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells C666-1 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ HSP70 Not detected contaminants Calnexin Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 13.54-225.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 80-100

	EV190099	1/3	Mus musculus	Serum	Filtration qEV	Anastasi, Federica	2020	50%
Study summary Full title Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model All authors Federica Anastasi, Francesco Greco, Marialaura Dilillo, Eleonora Vannini, Valentina Cappello, Laura Baroncelli, Mario Costa, Mauro Gemmi, Matteo Caleo & Liam A. McDonnell Journal Sci Rep Abstract Longitudinal analysis of disease models enables the molecular changes due to disease progression or (show more...)Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV’s) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV’s both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV’s isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1). (hide) EV-METRIC 50% (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 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 small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration qEV Protein markers EV: non-EV: Proteomics yes Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Sample Condition Control condition Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Proteomics Proteomics database No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 45

	EV190099	2/3	Mus musculus	Serum	Filtration Total Exosome Isolation	Anastasi, Federica	2020	50%
Study summary Full title Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model All authors Federica Anastasi, Francesco Greco, Marialaura Dilillo, Eleonora Vannini, Valentina Cappello, Laura Baroncelli, Mario Costa, Mauro Gemmi, Matteo Caleo & Liam A. McDonnell Journal Sci Rep Abstract Longitudinal analysis of disease models enables the molecular changes due to disease progression or (show more...)Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV’s) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV’s both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV’s isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1). (hide) EV-METRIC 50% (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 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 small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration Total Exosome Isolation Protein markers EV: non-EV: Proteomics yes Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Sample Condition Control condition Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method microBCA Proteomics Proteomics database No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 37

	EV190091	1/1	Homo sapiens	Cell culture supernatant	(d)(U)C SEC	Getnet Midekessa	2020	50%
Study summary Full title Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability All authors Getnet Midekessa, Kasun Godakumara, James Ord, Janeli Viil, Freddy Lättekivi, Keerthie Dissanayake, Sergei Kopanchuk, Ago Rinken, Aneta Andronowska, Sourav Bhattacharjee, Toonika Rinken, Alireza Fazeli Journal ACS Omega Abstract Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in i (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl2, and AlCl3) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant (p < 0.05) lowering of the ZP. Moreover, the ZP was significantly (p < 0.05) less negative in the presence of ions with higher valency (Al3+/Ca2+) than in the presence of monovalent ones (Na+/K+). Besides, the ZP of EVs became less negative at acidic pH, and vice versa. The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs. (hide) EV-METRIC 50% (80th 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 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 Cell Name JAR ATCC HTB-144 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. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C SEC Protein markers EV: CD81/ HSP70/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells JAR ATCC HTB-144 EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method Differential ultracentrifugation centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Size-exclusion chromatography Total column volume (mL) 10.5 Sample volume/column (mL) 0.5 Resin type Sepharose 4B Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ CD63/ HSP70/ CD81 Characterization: Particle analysis NTA Report type Mean Reported size (nm) 15 - 500 EV concentration Yes EM EM-type Transmission-EM/ Scanning-EM Image type Wide-field Report size (nm) 120 -200

	EV190075	1/3	Homo sapiens	Cell culture supernatant	Filtration Total Exosome Isolation UF	Thomas E. Whittaker	2020	50%
Study summary Full title Experimental artefacts can lead to misattribution

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