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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200080	1/4	Homo sapiens	Serum	(d)(U)C	Gabriella Dobra	2020	56%
Study summary Full title Small Extracellular Vesicles Isolated from Serum May Serve as Signal-Enhancers for the Monitoring of CNS Tumors All authors Gabriella Dobra, Matyas Bukva, Zoltan Szabo, Bella Bruszel, Maria Harmati, Edina Gyukity-Sebestyen, Adrienn Jenei, Monika Szucs, Peter Horvath, Tamas Biro, Almos Klekner, Krisztina Buzas Journal Int J Mol Sci Abstract Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may (show more...)Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may help to monitor brain tumors. In this proteomics-based study, we aimed to identify a characteristic protein fingerprint associated with central nervous system (CNS) tumors. Overall, 96 human serum samples were obtained from four patient groups, namely glioblastoma multiforme (GBM), non-small-cell lung cancer brain metastasis (BM), meningioma (M) and lumbar disc hernia patients (CTRL). After the isolation and characterization of small extracellular vesicles (sEVs) by nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM), liquid chromatography -mass spectrometry (LC-MS) was performed on two different sample types (whole serum and serum sEVs). Statistical analyses (ratio, Cohen's d, receiver operating characteristic; ROC) were carried out to compare patient groups. To recognize differences between the two sample types, pairwise comparisons (Welch's test) and ingenuity pathway analysis (IPA) were performed. According to our knowledge, this is the first study that compares the proteome of whole serum and serum-derived sEVs. From the 311 proteins identified, 10 whole serum proteins and 17 sEV proteins showed the highest intergroup differences. Sixty-five proteins were significantly enriched in sEV samples, while 129 proteins were significantly depleted compared to whole serum. Based on principal component analysis (PCA) analyses, sEVs are more suitable to discriminate between the patient groups. Our results support that sEVs have greater potential to monitor CNS tumors, than whole serum. (hide) EV-METRIC 56% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD81 non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type T-1270 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 111 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 9.29E+10 EM EM-type Atomic force-EM Image type Wide-field

	EV200080	2/4	Homo sapiens	Serum	(d)(U)C	Gabriella Dobra	2020	56%
Study summary Full title Small Extracellular Vesicles Isolated from Serum May Serve as Signal-Enhancers for the Monitoring of CNS Tumors All authors Gabriella Dobra, Matyas Bukva, Zoltan Szabo, Bella Bruszel, Maria Harmati, Edina Gyukity-Sebestyen, Adrienn Jenei, Monika Szucs, Peter Horvath, Tamas Biro, Almos Klekner, Krisztina Buzas Journal Int J Mol Sci Abstract Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may (show more...)Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may help to monitor brain tumors. In this proteomics-based study, we aimed to identify a characteristic protein fingerprint associated with central nervous system (CNS) tumors. Overall, 96 human serum samples were obtained from four patient groups, namely glioblastoma multiforme (GBM), non-small-cell lung cancer brain metastasis (BM), meningioma (M) and lumbar disc hernia patients (CTRL). After the isolation and characterization of small extracellular vesicles (sEVs) by nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM), liquid chromatography -mass spectrometry (LC-MS) was performed on two different sample types (whole serum and serum sEVs). Statistical analyses (ratio, Cohen's d, receiver operating characteristic; ROC) were carried out to compare patient groups. To recognize differences between the two sample types, pairwise comparisons (Welch's test) and ingenuity pathway analysis (IPA) were performed. According to our knowledge, this is the first study that compares the proteome of whole serum and serum-derived sEVs. From the 311 proteins identified, 10 whole serum proteins and 17 sEV proteins showed the highest intergroup differences. Sixty-five proteins were significantly enriched in sEV samples, while 129 proteins were significantly depleted compared to whole serum. Based on principal component analysis (PCA) analyses, sEVs are more suitable to discriminate between the patient groups. Our results support that sEVs have greater potential to monitor CNS tumors, than whole serum. (hide) EV-METRIC 56% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Glioblastoma multiforme Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD81 non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type T-1270 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 8.19E+10 EM EM-type Atomic force-EM Image type Wide-field

	EV200071	1/2	Homo sapiens	NPC/HK1	(d)(U)C 16% polyethylene glycol precipitation UF Filtration	Nkosi, Dingani	2020	56%
Study summary Full title Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells All authors Dingani Nkosi, Li Sun, Leanne C Duke, David G Meckes Jr Journal PLoS Pathog Abstract Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal ves (show more...)Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 16% polyethylene glycol precipitation UF Filtration Protein markers EV: TSG101/ CD63/ HSC70/ CD81/ Alix/ Flotillin2 non-EV: Calnexin Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NPC/HK1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Other Name other separation method 16% polyethylene glycol precipitation Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ HSC70/ Flotillin2/ TSG101/ Alix/ CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175.25 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3880000000000

	EV200071	2/2	Homo sapiens	NPC/HK1	(d)(U)C 16% polyethylene glycol precipitation UF Filtration	Nkosi, Dingani	2020	56%
Study summary Full title Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells All authors Dingani Nkosi, Li Sun, Leanne C Duke, David G Meckes Jr Journal PLoS Pathog Abstract Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal ves (show more...)Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin LMP1 overexpression Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 16% polyethylene glycol precipitation UF Filtration Protein markers EV: TSG101/ CD63/ HSC70/ CD81/ Alix/ Flotillin2 non-EV: Calnexin Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NPC/HK1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Other Name other separation method 16% polyethylene glycol precipitation Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Flotillin2/ HSC70/ TSG101/ Alix/ CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Other Reported size (nm) 186 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5300000000000

	EV200054	5/7	Mus musculus	Brain tissue	(d)(U)C Filtration qEV	Huang, Yiyao	2020	56%
Study summary Full title Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles All authors Yiyao Huang, Lesley Cheng, Andrey Turchinovich, Vasiliki Mahairaki, Juan C Troncoso, Olga Pletniková, Norman J Haughey, Laura J Vella, Andrew F Hill, Lei Zheng, Kenneth W Witwer Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processe (show more...)Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease. (hide) EV-METRIC 56% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Brain tissue Sample origin Control condition Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Commercial method Protein markers EV: Rab27a/ TSG101 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV- related methods Sample Species Mus musculus Sample Type Brain tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type AH-650 Pelleting: speed (g) 10000 Filtration steps 0.22µm or 0.2µm Commercial kit qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Rab27a Not detected EV-associated proteins TSG101 Detected contaminants GM130/ Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV200036	5/16	Homo sapiens	human skin primary fibroblasts	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Progeria patients Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Commercial method 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 EV-producing cells human skin primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 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 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GSTM2 Characterization: Lipid analysis No 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	primary human foreskin fibroblasts	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Commercial method 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 EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 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 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GSTM2 Characterization: Lipid analysis No 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	primary human foreskin fibroblasts	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin iRas Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Commercial method 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 EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 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 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GSTM2 Characterization: Lipid analysis No 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	primary human foreskin fibroblasts	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin iRas+GSTM2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Commercial method 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 EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 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 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GSTM2 Characterization: Lipid analysis No 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	primary human foreskin fibroblasts	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin iC+GSTM2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Commercial method 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 EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 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 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GSTM2 Characterization: Lipid analysis No 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	primary human foreskin fibroblasts	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin mCherry-CD63 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Commercial method 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 EV-producing cells primary human foreskin fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 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 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GSTM2 Characterization: Lipid analysis No 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	A549	(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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation 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 EV-producing cells A549 EV-harvesting Medium Serum free medium Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Not reported Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins TSG101/ CD9/ CD81/ CD63/ actin-beta Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type 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% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-80 Pelleting: speed (g) 100000 Other Name other separation method Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ Alix Not detected contaminants APOA1/ APOB100 Characterization: Lipid analysis No 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% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-80 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ Alix Not detected contaminants APOA1/ APOB100 Characterization: Lipid analysis No 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

	EV200019	1/4	Homo sapiens	Saliva	(d)(U)C Other;Lonza SEC column UF	Han, Pingping	2020	56%
Study summary Full title Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis All authors Pingping Han, Andrew Lai, Carlos Salomon, Sašo Ivanovski Journal Int J Mol Sci Abstract Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral disea (show more...)Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral diseases. However, technical difficulties for salivary sEV isolation remain a challenge. Twelve participants (five periodontally healthy, seven gingivitis patients) were recruited and salivary sEV were isolated by ultracentrifuge (UC-sEV) and size exclusion chromatography (SEC-sEV). The effect of UC and SEC on sEV yield, DNA methylation of five cytokine gene promoters (interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, IL-8, and IL-10), and functional uptake by human primary gingival fibroblasts (hGFs) was investigated. The results demonstrated that SEC-sEV had a higher yield of particles and particle/protein ratios compared to UC-sEV, with a minimal effect on the detection of DNA methylation of five cytokine genes and functional uptake in hGFs (n = 3). Comparing salivary sEV characteristics between gingivitis and healthy patients, gingivitis-UC-sEV were increased compared to the healthy group; while no differences were found in sEV size, oral bacterial gDNA, and DNA methylation for five cytokine gene promoters, for both UC-sEV and SEC-sEV. Overall, the data indicate that SEC results in a higher yield of salivary sEV, with no significant differences in sEV DNA epigenetics, compared to UC. (hide) EV-METRIC 56% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin Control condition Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Commercial method UF Protein markers EV: TSG101/ Alix/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 10000 Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Commercial kit Other;Lonza SEC column Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ Alix Characterization: RNA analysis RNA analysis Type Other Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Modus Reported size (nm) 145 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5.07E+10 EM EM-type Transmission-EM Image type Close-up

	EV200019	2/4	Homo sapiens	Saliva	(d)(U)C Other;Lonza SEC column UF	Han, Pingping	2020	56%
Study summary Full title Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis All authors Pingping Han, Andrew Lai, Carlos Salomon, Sašo Ivanovski Journal Int J Mol Sci Abstract Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral disea (show more...)Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral diseases. However, technical difficulties for salivary sEV isolation remain a challenge. Twelve participants (five periodontally healthy, seven gingivitis patients) were recruited and salivary sEV were isolated by ultracentrifuge (UC-sEV) and size exclusion chromatography (SEC-sEV). The effect of UC and SEC on sEV yield, DNA methylation of five cytokine gene promoters (interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, IL-8, and IL-10), and functional uptake by human primary gingival fibroblasts (hGFs) was investigated. The results demonstrated that SEC-sEV had a higher yield of particles and particle/protein ratios compared to UC-sEV, with a minimal effect on the detection of DNA methylation of five cytokine genes and functional uptake in hGFs (n = 3). Comparing salivary sEV characteristics between gingivitis and healthy patients, gingivitis-UC-sEV were increased compared to the healthy group; while no differences were found in sEV size, oral bacterial gDNA, and DNA methylation for five cytokine gene promoters, for both UC-sEV and SEC-sEV. Overall, the data indicate that SEC results in a higher yield of salivary sEV, with no significant differences in sEV DNA epigenetics, compared to UC. (hide) EV-METRIC 56% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin Control condition Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Commercial method UF Protein markers EV: TSG101/ Alix/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 10000 Ultra filtration Cut-off size (kDa) 10 Membrane type Not specified Commercial kit Other;Lonza SEC column Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ Alix Characterization: RNA analysis RNA analysis Type Other Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Modus Reported size (nm) 145 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.70E+11 EM EM-type Transmission-EM Image type Close-up

	EV200019	3/4	Homo sapiens	Saliva	(d)(U)C Other;Lonza SEC column	Han, Pingping	2020	56%
Study summary Full title Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis All authors Pingping Han, Andrew Lai, Carlos Salomon, Sašo Ivanovski Journal Int J Mol Sci Abstract Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral disea (show more...)Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral diseases. However, technical difficulties for salivary sEV isolation remain a challenge. Twelve participants (five periodontally healthy, seven gingivitis patients) were recruited and salivary sEV were isolated by ultracentrifuge (UC-sEV) and size exclusion chromatography (SEC-sEV). The effect of UC and SEC on sEV yield, DNA methylation of five cytokine gene promoters (interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, IL-8, and IL-10), and functional uptake by human primary gingival fibroblasts (hGFs) was investigated. The results demonstrated that SEC-sEV had a higher yield of particles and particle/protein ratios compared to UC-sEV, with a minimal effect on the detection of DNA methylation of five cytokine genes and functional uptake in hGFs (n = 3). Comparing salivary sEV characteristics between gingivitis and healthy patients, gingivitis-UC-sEV were increased compared to the healthy group; while no differences were found in sEV size, oral bacterial gDNA, and DNA methylation for five cytokine gene promoters, for both UC-sEV and SEC-sEV. Overall, the data indicate that SEC results in a higher yield of salivary sEV, with no significant differences in sEV DNA epigenetics, compared to UC. (hide) EV-METRIC 56% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin gingivitis Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Commercial method Protein markers EV: Alix/ TSG101/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 10000 Commercial kit Other;Lonza SEC column Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Modus Reported size (nm) 145 Particle yield NA NA EM EM-type Transmission-EM Image type Close-up

	EV200019	4/4	Homo sapiens	Saliva	(d)(U)C Other;Lonza SEC column	Han, Pingping	2020	56%
Study summary Full title Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis All authors Pingping Han, Andrew Lai, Carlos Salomon, Sašo Ivanovski Journal Int J Mol Sci Abstract Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral disea (show more...)Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral diseases. However, technical difficulties for salivary sEV isolation remain a challenge. Twelve participants (five periodontally healthy, seven gingivitis patients) were recruited and salivary sEV were isolated by ultracentrifuge (UC-sEV) and size exclusion chromatography (SEC-sEV). The effect of UC and SEC on sEV yield, DNA methylation of five cytokine gene promoters (interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, IL-8, and IL-10), and functional uptake by human primary gingival fibroblasts (hGFs) was investigated. The results demonstrated that SEC-sEV had a higher yield of particles and particle/protein ratios compared to UC-sEV, with a minimal effect on the detection of DNA methylation of five cytokine genes and functional uptake in hGFs (n = 3). Comparing salivary sEV characteristics between gingivitis and healthy patients, gingivitis-UC-sEV were increased compared to the healthy group; while no differences were found in sEV size, oral bacterial gDNA, and DNA methylation for five cytokine gene promoters, for both UC-sEV and SEC-sEV. Overall, the data indicate that SEC results in a higher yield of salivary sEV, with no significant differences in sEV DNA epigenetics, compared to UC. (hide) EV-METRIC 56% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin gingivitis Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Commercial method Protein markers EV: Alix/ TSG101/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 10000 Commercial kit Other;Lonza SEC column Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Not Reported EM EM-type Transmission-EM Image type Close-up

	EV200016	1/2	Bos taurus	Ovarian granulosa cells	(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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Commercial method 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 EV-producing cells Ovarian granulosa cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 75 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No 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	Ovarian granulosa cells	(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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Commercial method 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 EV-producing cells Ovarian granulosa cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 75 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected contaminants Cytochrome Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No 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	Primary cortical neurons	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture 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 EV-producing cells Primary cortical neurons EV-harvesting Medium Serum free medium Cell count 2,500,000 Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ syntenin Not detected EV-associated proteins tubulin Not detected contaminants gp96 Characterization: Lipid analysis No 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

	EV200008	1/18	Homo sapiens	Blood plasma	(d)(U)C	Peng, Cheng	2020	56%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 56% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 660 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 BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ vimentin/ TSG101/ CD81 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3070000000

	EV200008	10/18	Homo sapiens	Blood plasma	miRCURY Exosome Kit	Peng, Cheng	2020	56%
Study summary Full title Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples All authors Cheng Peng, Jizhuang Wang, Qiyuan Bao, Jun Wang, Zhuochao Liu, Junxiang Wen, Weibin Zhang, Yuhui Shen Journal Cancer Biomarkers Abstract Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although th (show more...)Background: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. Methods: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. Results: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. Conclusions: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research. (hide) EV-METRIC 56% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture miRCURY Exosome Kit Protein markers EV: TSG101/ CD81/ CD63/ CD9/ vimentin non-EV: calnexin/ Albumin/ Apolipoprotein A-1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit Other;miRCURY Exosome Kit Other Name other separation method miRCURY Exosome Kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ CD81 Not detected EV-associated proteins vimentin/ CD9 Detected contaminants Apolipoprotein A-1/ Albumin Not detected contaminants calnexin Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-240 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 56700000000

	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% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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. dUC = (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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes 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% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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. dUC = (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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes 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	Embryonic stem cells	(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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 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 EV-producing cells Embryonic stem cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected contaminants GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

	EV190069	3/4	Homo sapiens	DU145	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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. dUC = (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 EV-producing cells DU145 EV-harvesting Medium Serum free medium Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Density gradient 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins HSPA5 Not detected EV-associated proteins CD9 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 1650 EV concentration Yes

	EV190069	4/4	Homo sapiens	DU145	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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. dUC = (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 EV-producing cells DU145 EV-harvesting Medium Serum free medium Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Density gradient 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9 Not detected EV-associated proteins HSPA5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 116 EV concentration Yes

	EV190066	1/2	Homo sapiens	HEK	(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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 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 EV-producing cells HEK EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Surespin 630 (17 ml) Pelleting: speed (g) 28400 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Characterization: Lipid analysis No 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% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 41900 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101 Not detected EV-associated proteins TSG101/ CD63/ CD9/ Alix Characterization: Lipid analysis No 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% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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. dUC = (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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ CD9 Detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No 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% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD41/ TSG101 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD61/ CD235a/ phosphatidylserine Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Lipid analysis No 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% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD41/ TSG101 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD61/ CD235a/ phosphatidylserine Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Lipid analysis No 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% (88th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ CD41 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD61/ Cd235a/ phosphatidylserine Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Lipid analysis No 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% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD41/ CD9/ TSG101 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD41/ CD235a/ phosphatidylserine Proteomics database Yes: Other 1 Flow cytometry (after non-specific association of vesicles to beads) Characterization: Lipid analysis No 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	H9	(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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration 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 EV-producing cells H9 EV-harvesting Medium Serum free medium Cell viability (%) 100 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed 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 Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Other particle analysis name(1) qNano Report type Size range/distribution Report size 75-200 EV-concentration Yes

	EV230964	1/1	Homo sapiens	human milk	ExoQuick Filtration (d)(U)C	Bickmore DC	2020	50%
Study summary Full title Characterization of Extracellular Vesicles Isolated From Human Milk Using a Precipitation-Based Method. All authors Bickmore DC, Miklavcic JJ Journal Front Nutr Abstract Extracellular vesicles (EV) function in intercellular communication, and those in human milk may con (show more...)Extracellular vesicles (EV) function in intercellular communication, and those in human milk may confer immunologic benefits to infants. Methods of EV isolation such as ultracentrifugation (UC) may not be feasible for the study of EVs in human milk due to the need for large sample volume. A technique to isolate EVs from a small volume of human milk using a precipitation reagent is described herein. Electron microscopy, nanoparticle tracking analysis, and semi-quantitative antibody array were conducted to confirm isolation of human milk EVs. Count, size, protein content, and fatty acid quantification of EVs were determined. This isolation technique yielded 8.9 x 10 (± 1.1 × 10) EV particles/mL of human milk. The present method meets the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. An established EV isolation method suitable for a low volume of human milk will facilitate further research in this growing area. (hide) EV-METRIC 50% (25th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type human milk Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Filtration (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ anxa5/ EpCAM/ ICAM1 non-EV: GM130 Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type human milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps Between 0.22 and 0.45 µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ anxa5/ EpCAM/ ICAM1 Not detected contaminants GM130 Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 200-278 NTA Report type Modus Reported size (nm) 150 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.9E9 EM EM-type Scanning-EM Image type Wide-field Report size (nm) 50-350

	EV220244	1/4	Homo sapiens	Bone marrow-derived mesenchymal stem cells	(d)(U)C Total Exosome Isolation	Jiang L	2020	50%
Study summary Full title Exosomes derived from TSG-6 modified mesenchymal stromal cells attenuate scar formation during wound healing. All authors Jiang L, Zhang Y, Liu T, Wang X, Wang H, Song H, Wang W Journal Biochimie Abstract Mesenchymal stromal cell (MSC)-derived exosome therapy has emerged as an effective therapy strategy (show more...)Mesenchymal stromal cell (MSC)-derived exosome therapy has emerged as an effective therapy strategy for the pathological scar formation. However, the underlying mechanisms have not been completely understood. In the current study, we investigate the therapeutic effect of TSG-6 modified MSC-derived exosomes on a mouse full-thickness wound model and provide evidence of a possible mechanism for MSC-derived exosomes to prevent from scar formation. Overexpression and knockdown of TSG-6 were conducted by lentivirus infection into hBMSCs. Exosomes were isolated from cell culture and identified by transmission electron microscopy and Western blot. C57BL/6J mice were performed of full-thickness skin wounds and treated with exosomal suspension or TSG-6-neutralizing antibody. H&E staining was subjected to observe the pathological changes of scar tissues. Immunohistochemistry, ELISA, real time-PCR and Western blot were applied to detect the expressions of relevant molecules. The results showed that subcutaneous injection of TSG-6 overexpressed MSC-derived exosomes effectively ameliorated scar pathological injury, decreased inflammatory molecular secretion and attenuated collagen deposition in a mouse skin wound model. Reversely, knockdown of TSG-6 abrogated the therapeutic effect of MSC-derived exosomes on scarring. Moreover, TSG-6-neutralizing antibody counteracted the effect of TSG-6 overexpressed MSC-derived exosomes in preventing scar formation. In conclusion, we demonstrated that exosomes derived from TSG-6 modified MSCs suppressed scar formation via reducing inflammation and inhibiting collagen deposition. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD9/ CD63/ TSG101/ Alix/ TSG-6/ Actin beta non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Bone marrow-derived mesenchymal stem cells EV-harvesting Medium EV-depleted FBS Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ TSG-6/ Actin beta Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 20-100

	EV220079	1/3	Homo sapiens	adipose-derived stem cells	(d)(U)C UF Filtration	Jung YJ	2020	50%
Study summary Full title Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes. All authors Jung YJ, Kim HK, Cho Y, Choi JS, Woo CH, Lee KS, Sul JH, Lee CM, Han J, Park JH, Jo DG, Cho YW Journal Sci Adv Abstract Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based thera (show more...)Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based therapy for regenerative medicine. In this study, stem cell EVs derived during differentiation are developed to use as cell-free therapeutic systems by inducing tissue-specific differentiation. EVs are isolated from human adipose-derived stem cells (HASCs) during white and beige adipogenic differentiation (D-EV and BD-EV, respectively) via tangential flow filtration. D-EV and BD-EV can successfully differentiate HASCs into white and beige adipocytes, respectively. D-EV are transplanted with collagen/methylcellulose hydrogels on the backs of BALB/c mice, and they produce numerous lipid droplets in injected sites. Treatments of BD-EV attenuate diet-induced obesity through browning of adipose tissue in mice. Furthermore, high-fat diet-induced hepatic steatosis and glucose tolerance are improved by BD-EV treatment. miRNAs are responsible for the observed effects of BD-EV. These results reveal that secreted EVs during stem cell differentiation into white adipocytes or beige adipocytes can promote cell reprogramming. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: Alix/ TSG101/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adipose-derived stem cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 300 Membrane type NS Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 80 tot 600 NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Report size (nm) 80-120

	EV220079	2/3	Homo sapiens	adipose-derived stem cells	(d)(U)C UF Filtration	Jung YJ	2020	50%
Study summary Full title Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes. All authors Jung YJ, Kim HK, Cho Y, Choi JS, Woo CH, Lee KS, Sul JH, Lee CM, Han J, Park JH, Jo DG, Cho YW Journal Sci Adv Abstract Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based thera (show more...)Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based therapy for regenerative medicine. In this study, stem cell EVs derived during differentiation are developed to use as cell-free therapeutic systems by inducing tissue-specific differentiation. EVs are isolated from human adipose-derived stem cells (HASCs) during white and beige adipogenic differentiation (D-EV and BD-EV, respectively) via tangential flow filtration. D-EV and BD-EV can successfully differentiate HASCs into white and beige adipocytes, respectively. D-EV are transplanted with collagen/methylcellulose hydrogels on the backs of BALB/c mice, and they produce numerous lipid droplets in injected sites. Treatments of BD-EV attenuate diet-induced obesity through browning of adipose tissue in mice. Furthermore, high-fat diet-induced hepatic steatosis and glucose tolerance are improved by BD-EV treatment. miRNAs are responsible for the observed effects of BD-EV. These results reveal that secreted EVs during stem cell differentiation into white adipocytes or beige adipocytes can promote cell reprogramming. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin white adipogenic differentiation Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: Alix/ TSG101/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adipose-derived stem cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 300 Membrane type NS Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 100 tot 800 NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Report size (nm) 80-120

	EV220079	3/3	Homo sapiens	adipose-derived stem cells	(d)(U)C UF Filtration	Jung YJ	2020	50%
Study summary Full title Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes. All authors Jung YJ, Kim HK, Cho Y, Choi JS, Woo CH, Lee KS, Sul JH, Lee CM, Han J, Park JH, Jo DG, Cho YW Journal Sci Adv Abstract Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based thera (show more...)Stem cell-derived extracellular vesicles (EVs) offer alternative approaches to stem cell-based therapy for regenerative medicine. In this study, stem cell EVs derived during differentiation are developed to use as cell-free therapeutic systems by inducing tissue-specific differentiation. EVs are isolated from human adipose-derived stem cells (HASCs) during white and beige adipogenic differentiation (D-EV and BD-EV, respectively) via tangential flow filtration. D-EV and BD-EV can successfully differentiate HASCs into white and beige adipocytes, respectively. D-EV are transplanted with collagen/methylcellulose hydrogels on the backs of BALB/c mice, and they produce numerous lipid droplets in injected sites. Treatments of BD-EV attenuate diet-induced obesity through browning of adipose tissue in mice. Furthermore, high-fat diet-induced hepatic steatosis and glucose tolerance are improved by BD-EV treatment. miRNAs are responsible for the observed effects of BD-EV. These results reveal that secreted EVs during stem cell differentiation into white adipocytes or beige adipocytes can promote cell reprogramming. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin beige adipogenic differentiation Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: Alix/ TSG101/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adipose-derived stem cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 300 Membrane type NS Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 80 tot 800 NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Report size (nm) 80-120

	EV210377	4/8	Mus musculus	primary BM-MSCs	UF	Angioni R	2020	50%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration Protein markers EV: CD9/ CD63/ CD39/ CD73/ TIMP1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary BM-MSCs EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD39/ CD73/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV210377	6/8	Mus musculus	primary BM-MSCs	UF	Angioni R	2020	50%
Study summary Full title CD73 extracellular vesicles inhibit angiogenesis through adenosine A receptor signalling. All authors Angioni R, Liboni C, Herkenne S, Sánchez-Rodríguez R, Borile G, Marcuzzi E, Calì B, Muraca M, Viola A Journal J Extracell Vesicles Abstract Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory d (show more...)Pathological angiogenesis is a hallmark of several conditions including eye diseases, inflammatory diseases, and cancer. Stromal cells play a crucial role in regulating angiogenesis through the release of soluble factors or direct contact with endothelial cells. Here, we analysed the properties of the extracellular vesicles (EVs) released by bone marrow mesenchymal stromal cells (MSCs) and explored the possibility of using them to therapeutically target angiogenesis. We demonstrated that in response to pro-inflammatory cytokines, MSCs produce EVs that are enriched in TIMP-1, CD39 and CD73 and inhibit angiogenesis targeting both extracellular matrix remodelling and endothelial cell migration. We identified a novel anti-angiogenic mechanism based on adenosine production, triggering of A adenosine receptors, and induction of NOX2-dependent oxidative stress within endothelial cells. Finally, in pilot experiments, we exploited the anti-angiogenic EVs to inhibit tumour progression . Our results identify novel pathways involved in the crosstalk between endothelial and stromal cell and suggest new therapeutic strategies to target pathological angiogenesis. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Cytokines-treated Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration Protein markers EV: CD9/ CD63/ TIMP1/ CD39/ CD73 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary BM-MSCs EV-harvesting Medium Serum free medium Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TIMP1/ CD39/ CD73 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes EM EM-type Transmission?-EM Image type Close-up

	EV210348	1/6	Homo sapiens	Serum	ExoQuick	Arya R	2020	50%
Study summary Full title Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response. All authors Arya R, Dabral D, Faruquee HM, Mazumdar H, Patgiri SJ, Deka T, Basumatary R, Kupa RU, Semy C, Kapfo W, Liegise K, Kaur I, Choedon T, Kumar P, Behera RK, Deori P, Nath R, Khalo K, Saikia L, Khamo V, Nanda RK Journal Proteomics Clin Appl Abstract Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for ident (show more...)Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. (hide) EV-METRIC 50% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Protein markers EV: SPON2/ AQP2/ CD63/ Alix/ NAPSA/ CD9/ KYAT3/ SERPINA1/ HP/ APOC3 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ AQP2/ SPON2/ NAPSA/ Alix/ KYAT3/ SERPINA1/ HP/ APOC3 Flow cytometry Type of Flow cytometry BD FACSCanto II Antibody details provided? No Detected EV-associated proteins CD9 Proteomics database Yes: ProteomeXchange Consortiu Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution EM EM-type Immuno-EM EM protein CD9 Image type Close-up Report size (nm) 20-200

	EV210348	3/6	Homo sapiens	Serum	ExoQuick	Arya R	2020	50%
Study summary Full title Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response. All authors Arya R, Dabral D, Faruquee HM, Mazumdar H, Patgiri SJ, Deka T, Basumatary R, Kupa RU, Semy C, Kapfo W, Liegise K, Kaur I, Choedon T, Kumar P, Behera RK, Deori P, Nath R, Khalo K, Saikia L, Khamo V, Nanda RK Journal Proteomics Clin Appl Abstract Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for ident (show more...)Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. (hide) EV-METRIC 50% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Drug naive active tuberculosis Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Protein markers EV: AQP2/ SPON2/ NAPSA/ Alix/ CD63/ CD9/ KYAT3/ SERPINA1/ HP/ APOC3 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63/ NAPSA/ SPON2/ AQP2/ KYAT3/ SERPINA1/ HP/ APOC3 Flow cytometry Type of Flow cytometry BD FACSCanto II Antibody details provided? No Detected EV-associated proteins CD9 Proteomics database Yes: ProteomeXchange Consortiu Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution EM EM-type Immuno-EM EM protein CD9 Image type Close-up Report size (nm) 20-200

	EV210348	5/6	Homo sapiens	Serum	ExoQuick	Arya R	2020	50%
Study summary Full title Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response. All authors Arya R, Dabral D, Faruquee HM, Mazumdar H, Patgiri SJ, Deka T, Basumatary R, Kupa RU, Semy C, Kapfo W, Liegise K, Kaur I, Choedon T, Kumar P, Behera RK, Deori P, Nath R, Khalo K, Saikia L, Khamo V, Nanda RK Journal Proteomics Clin Appl Abstract Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for ident (show more...)Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. (hide) EV-METRIC 50% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Non-tuberculosis Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Protein markers EV: SPON2/ AQP2/ Alix/ CD63/ CD9/ NAPSA/ KYAT3/ SERPINA1/ HP/ APOC3 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins NAPSA/ SPON2/ AQP2/ CD9/ CD63/ Alix/ KYAT3/ SERPINA1/ HP/ APOC3 Flow cytometry Type of Flow cytometry BD FACSCanto II Antibody details provided? No Detected EV-associated proteins CD9 Proteomics database Yes: ProteomeXchange Consortiu Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution EM EM-type Immuno-EM EM protein CD9 Image type Close-up Report size (nm) 20-200

	EV210222	1/2	Homo sapiens	Blood plasma	ExoQuick	Monteiro, Lara J	2020	50%
Study summary Full title Increased Circulating Levels of Tissue-Type Plasminogen Activator Are Associated with the Risk of Spontaneous Abortion During the First Trimester of Pregnancy All authors Lara J Monteiro, Manuel Varas-Godoy, Stephanie Acuña-Gallardo, Paula Correa, Gianluca Passalacqua, Max Monckeberg, Gregory E Rice, Sebastián E Illanes Journal Diagnostics Abstract Spontaneous abortion is a common complication in early pregnancy, with an incidence of around 20%. U (show more...)Spontaneous abortion is a common complication in early pregnancy, with an incidence of around 20%. Ultrasound scan and measurement of human chorionic gonadotropin are used to identify patients at risk of spontaneous abortion; however, there is a clinical need to find new biomarkers to prospectively identify patients before the onset of clinical symptoms. Here, we aim to investigate potential biomarkers of spontaneous abortion taken in the first clinical appointment of pregnancy. A case-control study was conducted based on a prospectively collected cohort in which cases and controls were retrospectively stratified based on pregnancy outcome: normal healthy pregnancies (controls = 33) and pregnancies that ended in spontaneous abortion (cases = 10). We evaluated extracellular vesicles isolated by precipitation with ExoQuick™ and protein concentrations of tissue plasminogen activator, leptin, and adiponectin measured by ELISA. The extracellular vesicles showed the typical morphology and membrane proteins: CD63, Alix, and Flotilin-1. The size distributions of the isolated extracellular vesicles were 112 ± 27 and 118 ± 28 nm in diameter for controls and spontaneous abortion, respectively, and the total amount of extracellular vesicles did not show any difference between controls and the spontaneous abortion group. The tissue plasminogen activator showed a significant difference (p = 0.0004) between both groups, although neither adiponectin nor leptin revealed significant changes, indicating that women who had spontaneous abortions have significantly higher levels of tissue plasminogen activator than women who had normal pregnancies. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Healthy pregnant Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Protein markers EV: Alix/ CD63/ Flotillin1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD63/ Alix Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 112 +/- 27 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6e11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210222	2/2	Homo sapiens	Blood plasma	ExoQuick	Monteiro, Lara J	2020	50%
Study summary Full title Increased Circulating Levels of Tissue-Type Plasminogen Activator Are Associated with the Risk of Spontaneous Abortion During the First Trimester of Pregnancy All authors Lara J Monteiro, Manuel Varas-Godoy, Stephanie Acuña-Gallardo, Paula Correa, Gianluca Passalacqua, Max Monckeberg, Gregory E Rice, Sebastián E Illanes Journal Diagnostics Abstract Spontaneous abortion is a common complication in early pregnancy, with an incidence of around 20%. U (show more...)Spontaneous abortion is a common complication in early pregnancy, with an incidence of around 20%. Ultrasound scan and measurement of human chorionic gonadotropin are used to identify patients at risk of spontaneous abortion; however, there is a clinical need to find new biomarkers to prospectively identify patients before the onset of clinical symptoms. Here, we aim to investigate potential biomarkers of spontaneous abortion taken in the first clinical appointment of pregnancy. A case-control study was conducted based on a prospectively collected cohort in which cases and controls were retrospectively stratified based on pregnancy outcome: normal healthy pregnancies (controls = 33) and pregnancies that ended in spontaneous abortion (cases = 10). We evaluated extracellular vesicles isolated by precipitation with ExoQuick™ and protein concentrations of tissue plasminogen activator, leptin, and adiponectin measured by ELISA. The extracellular vesicles showed the typical morphology and membrane proteins: CD63, Alix, and Flotilin-1. The size distributions of the isolated extracellular vesicles were 112 ± 27 and 118 ± 28 nm in diameter for controls and spontaneous abortion, respectively, and the total amount of extracellular vesicles did not show any difference between controls and the spontaneous abortion group. The tissue plasminogen activator showed a significant difference (p = 0.0004) between both groups, although neither adiponectin nor leptin revealed significant changes, indicating that women who had spontaneous abortions have significantly higher levels of tissue plasminogen activator than women who had normal pregnancies. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Pregnant with later spontaneous abortion Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Protein markers EV: Alix/ CD63/ Flotillin1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD63/ Alix Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 118 +/- 28 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5.5e11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210184	2/4	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Pillay, Preenan	2020	50%
Study summary Full title Exosomal Th1/Th2 cytokines in preeclampsia and HIV-positive preeclamptic women on highly active anti-retroviral therapy All authors Preenan Pillay, Kogi Moodley, Manu Vatish, Jagidesa Moodley, Raquel Duarte, Irene Mackraj Journal Cytokine Abstract Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and f (show more...)Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and foetal morbidity and mortality. Furthermore, HIV/Highly Active Anti-Retroviral Treatment has been associated with the increased risk of preeclampsia due to maternal immune reconstitution, which complicates the clinical diagnosis of PE in these patients. It is therefore necessary to identify biomarkers involved in the pathology of both disorders with the intent to diagnose. Exosomal cytokines represent ideal biomarkers of PE and inflammatory conditions due to their immunomodulatory role in pregnancy. We therefore quantified exosomal Th1 (IL-2 and TNF-α) and Th2 cytokines (IL-10) in maternal circulation. A significant dysregulation in total exosomes, placental-derived exosomes and exosomal cytokines in PE and HIV-positive PE pregnant woman on Highly Active Antiretroviral Treatment (HAART) was observed (p < 0.01). Additionally, we observed a significant shift towards Th1 immunity in PE which becomes amplified in HIV-positive PE pregnant woman on HAART (p < 0.01). Moreover, we show the potential application of exosomal Tumor necrosis factor alpha (TNF-α) as a biomarker of PE and PE in HIV-positive pregnant women on HAART (CI: 95%, LHR > 10, sensitivity of 100% and specificity of 90%). These findings are in support of exosome release and exosome cytokine encapsulation as a tightly regulated process in favour of maintaining the immune microenvironment, which can orchestrate either normal pregnancy, or the pathogenesis of preeclampsia and preeclampsia in HIV/HAART pregnancies. (hide) EV-METRIC 50% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Normotensive pregnant with HIV Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Protein markers EV: PLAP/ CD63/ IL-2/ IL-10/ TNF-? non-EV: None Proteomics no EV density (g/ml) Not specified Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-55 Pelleting: speed (g) 110000 Density gradient Type Continuous Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Not specified Rotor type MLA-55 Speed (g) 200000 Duration (min) 120 Fraction volume (mL) 12 Fraction processing Not specified Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins CD63 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ PLAP Other 1 Multiplex cytokine assay Detected EV-associated proteins IL-2/ IL-10/ TNF-? Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 20-130nm EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 9.96e9 (< 33 gest weeks), 1.22e10 (>34 gest weeks) EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-130nm

	EV200117	2/6	Homo sapiens	HEK	(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% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin HEK MOCK Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration Commercial method 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 EV-producing cells HEK EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 89 Cell count 3.5E7-8.5E7 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ syntenin-1/ ACE2/ ADAM10/ CD81/ HSP70 Not detected contaminants AChe ELISA Antibody details provided? No Detected EV-associated proteins ACE2 Flow cytometry Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ syntenin-1 Detected EV-associated proteins CD81/ CD63/ syntenin-1 Characterization: Lipid analysis No 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

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