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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210493	3/4	Homo sapiens	hCMEC/D3	(d)(U)C	Dozio V	2017	29%
Study summary Full title Characterisation of extracellular vesicle-subsets derived from brain endothelial cells and analysis of their protein cargo modulation after TNF exposure. All authors Dozio V, Sanchez JC Journal J Extracell Vesicles Abstract Little is known about the composition and functional differences between extracellular vesicle (EV) (show more...)Little is known about the composition and functional differences between extracellular vesicle (EV) subsets, such as microvesicles (MVs) and exosomes (EXOs), nor to what extent their cargo reflects the phenotypic state of the cell of origin. Brain endothelial cells are the constitutive part of the blood-brain barrier (BBB), a selective barrier that maintains brain homeostasis. BBB impairment is associated with several neuroinflammatory diseases with the pro-inflammatory cytokine tumour necrosis factor (TNF) often playing a key role. In the present study, shotgun proteomics and parallel reaction monitoring (PRM)-based targeted mass spectrometry were used to characterise brain endothelial cell-released EVs, and to study how TNF exposure modulated EV protein cargoes. MVs were found to be enriched in mitochondrial and cytoskeletal proteins, whereas EXOs were enriched in adhesion, histone and ribosomal proteins. After stimulation with TNF, several proteins involved in TNF and NF-κB signalling pathways, that were found to be differentially expressed in cells, were also differentially expressed in both MVs and EXOs. Thus, our results revealed some novel proteins as potentially useful candidates for discriminating between MVs and EXOs, together with additional evidence that cells "package" proteins in EVs systematically and according to their phenotypic state. (hide) EV-METRIC 29% (68th 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 TNF Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hCMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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: rotor type SW 32 Ti Pelleting: speed (g) 18000 Wash: time (min) 30 Wash: Rotor Type SW 55 Ti Wash: speed (g) 18000 Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No

	EV210493	4/4	Homo sapiens	hCMEC/D3	(d)(U)C	Dozio V	2017	29%
Study summary Full title Characterisation of extracellular vesicle-subsets derived from brain endothelial cells and analysis of their protein cargo modulation after TNF exposure. All authors Dozio V, Sanchez JC Journal J Extracell Vesicles Abstract Little is known about the composition and functional differences between extracellular vesicle (EV) (show more...)Little is known about the composition and functional differences between extracellular vesicle (EV) subsets, such as microvesicles (MVs) and exosomes (EXOs), nor to what extent their cargo reflects the phenotypic state of the cell of origin. Brain endothelial cells are the constitutive part of the blood-brain barrier (BBB), a selective barrier that maintains brain homeostasis. BBB impairment is associated with several neuroinflammatory diseases with the pro-inflammatory cytokine tumour necrosis factor (TNF) often playing a key role. In the present study, shotgun proteomics and parallel reaction monitoring (PRM)-based targeted mass spectrometry were used to characterise brain endothelial cell-released EVs, and to study how TNF exposure modulated EV protein cargoes. MVs were found to be enriched in mitochondrial and cytoskeletal proteins, whereas EXOs were enriched in adhesion, histone and ribosomal proteins. After stimulation with TNF, several proteins involved in TNF and NF-κB signalling pathways, that were found to be differentially expressed in cells, were also differentially expressed in both MVs and EXOs. Thus, our results revealed some novel proteins as potentially useful candidates for discriminating between MVs and EXOs, together with additional evidence that cells "package" proteins in EVs systematically and according to their phenotypic state. (hide) EV-METRIC 29% (68th 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 TNF Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hCMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No

	EV210245	1/4	Homo sapiens	Jurkat	(d)(U)C	Mihály J	2017	29%
Study summary Full title Characterization of extracellular vesicles by IR spectroscopy: Fast and simple classification based on amide and CH stretching vibrations. All authors Mihály J, Deák R, Szigyártó IC, Bóta A, Beke-Somfai T, Varga Z Journal Biochim Biophys Acta Biomembr Abstract Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investig (show more...)Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investigated by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). Amide and CH stretching band intensity ratios calculated from IR bands, characteristic of protein and lipid components, proved to be distinctive for the different extracellular vesicle subpopulations. This proposed 'spectroscopic protein-to-lipid ratio', combined with the outlined spectrum-analysis protocol is valid also for low sample concentrations (0.15-0.05mg/ml total protein content) and can carry information about the presence of other non-vesicular formations such as aggregated proteins, lipoproteins and immune complexes. Detailed analysis of IR data reveals compositional changes of extracellular vesicles subpopulations: second derivative spectra suggest changes in protein composition from parent cell towards exosomes favoring proteins with β-turns and unordered motifs at the expense of intermolecular β-sheet structures. The IR-based protein-to-lipid assessment protocol was tested also for red blood cell derived microvesicles for which similar values were obtained. The potential applicability of this technique for fast and efficient characterization of vesicular components is high as the investigated samples require no further preparations and all the different molecular species can be determined in the same sample. The results indicate that ATR-FTIR measurements provide a simple and reproducible method for the screening of extracellular vesicle preparations. It is hoped that this sophisticated technique will have further impact in extracellular vesicle research. (hide) EV-METRIC 29% (68th 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium Serum free medium 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 T-1270 Pelleting: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Bradford Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean EM EM-type Transmission-EM Image type Close-up Report size (nm) 100

	EV210245	2/4	Homo sapiens	Jurkat	(d)(U)C	Mihály J	2017	29%
Study summary Full title Characterization of extracellular vesicles by IR spectroscopy: Fast and simple classification based on amide and CH stretching vibrations. All authors Mihály J, Deák R, Szigyártó IC, Bóta A, Beke-Somfai T, Varga Z Journal Biochim Biophys Acta Biomembr Abstract Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investig (show more...)Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investigated by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). Amide and CH stretching band intensity ratios calculated from IR bands, characteristic of protein and lipid components, proved to be distinctive for the different extracellular vesicle subpopulations. This proposed 'spectroscopic protein-to-lipid ratio', combined with the outlined spectrum-analysis protocol is valid also for low sample concentrations (0.15-0.05mg/ml total protein content) and can carry information about the presence of other non-vesicular formations such as aggregated proteins, lipoproteins and immune complexes. Detailed analysis of IR data reveals compositional changes of extracellular vesicles subpopulations: second derivative spectra suggest changes in protein composition from parent cell towards exosomes favoring proteins with β-turns and unordered motifs at the expense of intermolecular β-sheet structures. The IR-based protein-to-lipid assessment protocol was tested also for red blood cell derived microvesicles for which similar values were obtained. The potential applicability of this technique for fast and efficient characterization of vesicular components is high as the investigated samples require no further preparations and all the different molecular species can be determined in the same sample. The results indicate that ATR-FTIR measurements provide a simple and reproducible method for the screening of extracellular vesicle preparations. It is hoped that this sophisticated technique will have further impact in extracellular vesicle research. (hide) EV-METRIC 29% (68th 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 (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium Serum free medium 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 T-1270 Pelleting: speed (g) 20000 Characterization: Protein analysis Protein Concentration Method Bradford Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean EM EM-type Transmission-EM Image type Close-up Report size (nm) 200

	EV210245	3/4	Homo sapiens	Erythrocytes	(d)(U)C	Mihály J	2017	29%
Study summary Full title Characterization of extracellular vesicles by IR spectroscopy: Fast and simple classification based on amide and CH stretching vibrations. All authors Mihály J, Deák R, Szigyártó IC, Bóta A, Beke-Somfai T, Varga Z Journal Biochim Biophys Acta Biomembr Abstract Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investig (show more...)Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investigated by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). Amide and CH stretching band intensity ratios calculated from IR bands, characteristic of protein and lipid components, proved to be distinctive for the different extracellular vesicle subpopulations. This proposed 'spectroscopic protein-to-lipid ratio', combined with the outlined spectrum-analysis protocol is valid also for low sample concentrations (0.15-0.05mg/ml total protein content) and can carry information about the presence of other non-vesicular formations such as aggregated proteins, lipoproteins and immune complexes. Detailed analysis of IR data reveals compositional changes of extracellular vesicles subpopulations: second derivative spectra suggest changes in protein composition from parent cell towards exosomes favoring proteins with β-turns and unordered motifs at the expense of intermolecular β-sheet structures. The IR-based protein-to-lipid assessment protocol was tested also for red blood cell derived microvesicles for which similar values were obtained. The potential applicability of this technique for fast and efficient characterization of vesicular components is high as the investigated samples require no further preparations and all the different molecular species can be determined in the same sample. The results indicate that ATR-FTIR measurements provide a simple and reproducible method for the screening of extracellular vesicle preparations. It is hoped that this sophisticated technique will have further impact in extracellular vesicle research. (hide) EV-METRIC 29% (68th 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 apoptotic bodies Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Erythrocytes EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type T-1270 Pelleting: speed (g) 3000 Characterization: Protein analysis Protein Concentration Method Bradford Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean EM EM-type Transmission-EM Image type Close-up Report size (nm) 1000

	EV170000	1/2	Mus musculus	Serum	(d)(U)C Filtration	Thomou T	2017	29%
Study summary Full title Adipose-derived circulating miRNAs regulate gene expression in other tissues. All authors Thomou T, Mori MA, Dreyfuss JM, Konishi M, Sakaguchi M, Wolfrum C, Rao TN, Winnay JN, Garcia-Martin R, Grinspoon SK, Gorden P, Kahn Journal Nature Abstract Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism thro (show more...)Adipose tissue is a major site of energy storage and has a role in the regulation of metabolism through the release of adipokines. Here we show that mice with an adipose-tissue-specific knockout of the microRNA (miRNA)-processing enzyme Dicer (ADicerKO), as well as humans with lipodystrophy, exhibit a substantial decrease in levels of circulating exosomal miRNAs. Transplantation of both white and brown adipose tissue-brown especially-into ADicerKO mice restores the level of numerous circulating miRNAs that are associated with an improvement in glucose tolerance and a reduction in hepatic Fgf21 mRNA and circulating FGF21. This gene regulation can be mimicked by the administration of normal, but not ADicerKO, serum exosomes. Expression of a human-specific miRNA in the brown adipose tissue of one mouse in vivo can also regulate its 3' UTR reporter in the liver of another mouse through serum exosomal transfer. Thus, adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues and thereby serve as a previously undescribed form of adipokine. (hide) EV-METRIC 29% (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 Serum Sample origin NA 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 (d)(U)C Filtration Protein markers EV: CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus 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) 60 Pelleting: speed (g) 100000 Wash: time (min) 60 Wash: speed (g) 100000 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution EM EM-type Transmission-EM/ Immune-EM EM protein CD63;CD9 Image type Close-up, Wide-field Report size (nm) 80-200 Other particle analysis name(1) EXOCET ELISA assay EV-concentration Yes

	EV170036	8/12	Homo sapiens	Serum	(d)(U)C Filtration	Krafft C	2017	28%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 28% (70th 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 Benign prostate hyperplasia Focus vesicles EV12 Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 213.2 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes we proceeded with IR and RAMAN analysis of EVs isolated by 12000 x g (frequently designated as micro Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) 4-6 for healty donors in EV12; 100 in cancer patients; Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-200 Particle yield 1.10E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170036	10/12	Homo sapiens	Serum	(d)(U)C Filtration	Krafft C	2017	28%
Study summary Full title A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening. All authors Krafft C, Wilhelm K, Eremin A, Nestel S, von Bubnoff N, Schultze-Seemann W, Popp J, Nazarenko I Journal J Cell Sci Abstract In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and syste (show more...)In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia. (hide) EV-METRIC 28% (70th 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 Prostate cancer Focus vesicles EV120 Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 213.2 (pelleting) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes we proceeded with IR and RAMAN analysis of EVs isolated by 12000 x g (frequently designated as micro Characterization: Protein analysis None Protein Concentration Method microBCA Protein Yield (µg) 4-6 for healty donors in EV12; 100 in cancer patients; Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-200 Particle yield 1.00E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Other particle analysis name(1) Raman spectroscopy

	EV170005	1/4	Homo sapiens	SK-MEL103	(d)(U)C SEC UF	Suárez H	2017	28%
Study summary Full title A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles. All authors Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M Journal Sci Rep Abstract Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide) EV-METRIC 28% (65th 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 SEC UF Protein markers EV: CD81/ CD59/ CD63/ CD9/ MHC1 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL103 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) Not spec Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 148.16 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median

	EV170005	4/4	Homo sapiens	Primary T-lymphoblasts	(d)(U)C SEC UF	Suárez H	2017	28%
Study summary Full title A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles. All authors Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M Journal Sci Rep Abstract Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide) EV-METRIC 28% (65th 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 SEC UF Protein markers EV: CD81/ CD59/ CD63/ CD9/ MHC1 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary T-lymphoblasts EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) Not spec Membrane type Not specified Size-exclusion chromatography Total column volume (mL) 20 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 116.85 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median

	EV220426	2/2	Homo sapiens	urine	(d)(U)C ME-kit UF	Bijnsdorp IV	2017	25%
Study summary Full title Feasibility of urinary extracellular vesicle proteome profiling using a robust and simple, clinically applicable isolation method. All authors Bijnsdorp IV, Maxouri O, Kardar A, Schelfhorst T, Piersma SR, Pham TV, Vis A, van Moorselaar RJ, Jimenez CR Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) secreted by prostate cancer (PCa) cells contain specific biomarkers and (show more...)Extracellular vesicles (EVs) secreted by prostate cancer (PCa) cells contain specific biomarkers and can be isolated from urine. Collection of urine is not invasive, and therefore urinary EVs represent a liquid biopsy for diagnostic and prognostic testing for PCa. In this study, we optimised urinary EV isolation using a method based on heat shock proteins and compared it to gold-standard ultracentrifugation. The urinary EV isolation protocol using the Vn96-peptide is easier, time convenient (≈1.5 h) and no special equipment is needed, in contrast to ultracentrifugation protocol (>3.5 h), making this protocol clinically feasible. We compared the isolated vesicles of both ultracentrifugation and Vn96-peptide by proteome profiling using mass spectrometry-based proteomics ( = 4 per method). We reached a depth of >3000 proteins, with 2400 proteins that were commonly detected in urinary EVs from different donors. We show a large overlap (>85%) between proteins identified in EVs isolated by ultracentrifugation and Vn96-peptide. Addition of the detergent NP40 to Vn96-peptide EV isolations reduced levels of background proteins and highly increased the levels of the EV-markers TSG101 and PDCD6IP, indicative of an increased EV yield. Thus, the Vn96-peptide-based EV isolation procedure is clinically feasibly and allows large-scale protein profiling of urinary EV biomarkers. (hide) EV-METRIC 25% (56th 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 (Differential) (ultra)centrifugation Commercial method Ultrafiltration Protein markers EV: TSG101/ Alix non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type urine 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 No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit ME-kit Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins TSG101/ Alix Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220185	3/10	Homo sapiens	LNCaP	(d)(U)C Filtration IAF	Szczepanek D	2017	25%
Study summary Full title Primary central nervous system lymphoma as a neurosurgical problem. All authors Szczepanek D, Wąsik-Szczepanek E, Stoma F, Sokołowska B, Trojanowski T Journal BMC Cancer Abstract Primary central nervous system lymphoma (PCNSL) comprises around 3-5% of primary central nervous sys (show more...)Primary central nervous system lymphoma (PCNSL) comprises around 3-5% of primary central nervous system (CNS) tumours and around 1% of all non-Hodgkin lymphoma (NHL). Diffuse large B-cell lymphoma (DLBCL) is the most common histological type. High effectiveness of chemo- and radiotherapy for PCNSL regrettably does not eliminate significant risks of recurrence for CNS tumours. That results in higher interest in other treatment options, including surgical procedures. PCNSL remains in the scope of interest for many specialists and neurosurgeons seem to play a more important role. (hide) EV-METRIC 25% (64th 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 Filtration Immunoaffinity capture (non-commercial) Protein markers EV: Alix/ CD9/ GGT1/ PSMA non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium 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 0.2 or 0.22 µm Immunoaffinity capture Selected surface protein(s) CD9/ PSMA Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ GGT1/ PSMA Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220185	4/10	Homo sapiens	C4	(d)(U)C Filtration IAF	Szczepanek D	2017	25%
Study summary Full title Primary central nervous system lymphoma as a neurosurgical problem. All authors Szczepanek D, Wąsik-Szczepanek E, Stoma F, Sokołowska B, Trojanowski T Journal BMC Cancer Abstract Primary central nervous system lymphoma (PCNSL) comprises around 3-5% of primary central nervous sys (show more...)Primary central nervous system lymphoma (PCNSL) comprises around 3-5% of primary central nervous system (CNS) tumours and around 1% of all non-Hodgkin lymphoma (NHL). Diffuse large B-cell lymphoma (DLBCL) is the most common histological type. High effectiveness of chemo- and radiotherapy for PCNSL regrettably does not eliminate significant risks of recurrence for CNS tumours. That results in higher interest in other treatment options, including surgical procedures. PCNSL remains in the scope of interest for many specialists and neurosurgeons seem to play a more important role. (hide) EV-METRIC 25% (64th 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 Filtration Immunoaffinity capture (non-commercial) Protein markers EV: Alix/ CD9/ GGT1/ PSMA non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells C4 EV-harvesting Medium EV-depleted medium 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 0.2 or 0.22 µm Immunoaffinity capture Selected surface protein(s) CD9/ PSMA Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ GGT1/ PSMA Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220185	10/10	Homo sapiens	Serum	EVSecond	Szczepanek D	2017	25%
Study summary Full title Primary central nervous system lymphoma as a neurosurgical problem. All authors Szczepanek D, Wąsik-Szczepanek E, Stoma F, Sokołowska B, Trojanowski T Journal BMC Cancer Abstract Primary central nervous system lymphoma (PCNSL) comprises around 3-5% of primary central nervous sys (show more...)Primary central nervous system lymphoma (PCNSL) comprises around 3-5% of primary central nervous system (CNS) tumours and around 1% of all non-Hodgkin lymphoma (NHL). Diffuse large B-cell lymphoma (DLBCL) is the most common histological type. High effectiveness of chemo- and radiotherapy for PCNSL regrettably does not eliminate significant risks of recurrence for CNS tumours. That results in higher interest in other treatment options, including surgical procedures. PCNSL remains in the scope of interest for many specialists and neurosurgeons seem to play a more important role. (hide) EV-METRIC 25% (67th 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 Prostate Cancer Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture EVSecond Protein markers EV: CD9/ GGT1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit EVSecond Other Name other separation method EVSecond Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9/ GGT1 ELISA Antibody details provided? No Detected EV-associated proteins CD9 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220184	3/5	Homo sapiens	Blood plasma	(d)(U)C Filtration	Logozzi M	2017	25%
Study summary Full title Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients. All authors Logozzi M, Angelini DF, Iessi E, Mizzoni D, Di Raimo R, Federici C, Lugini L, Borsellino G, Gentilucci A, Pierella F, Marzio V, Sciarra A, Battistini L, Fais S Journal Cancer Lett Abstract Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis (show more...)Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis of prostate cancer (PCa). While neoplastic lesions of the prostate may cause aberrant levels of PSA in the blood, the quantitation of free or complexed PSA poorly discriminates cancer patients from those developing benign lesions, often leading to invasive and unnecessary surgical procedures. Microenvironmental acidity increases exosome release by cancer cells. In this study we evaluated whether acidity, a critical phenotype of malignancy, could influence exosome release and increase the PSA expression in nanovesicles released by PCa cells. To this aim, we exploited Nanoparticle Tracking Analysis (NTA), an immunocapture-based ELISA, and nanoscale flow-cytometry. The results show that microenvironmental acidity induces an increased release of nanovesicles expressing both PSA and the exosome marker CD81. In order to verify whether the changes induced by the local selective pressure of extracellular acidity may correspond to a clinical pathway we used the same approach to evaluate the levels of PSA-expressing exosomes in the plasma of PCa patients and controls, including subjects with benign prostatic hypertrophy (BPH). The results show that only PCa patients have high levels of nanovesicles expressing both CD81 and PSA. This study shows that tumor acidity exerts a selective pressure leading to the release of extracellular vesicles that express both PSA and exosome markers. A comparable scenario was shown in the plasma of prostate cancer patients as compared to both BPH and healthy controls. These results suggest that microenvironmental acidity may represent a key factor which determines qualitatively and quantitatively the release of extracellular vesicles by malignant tumors, including prostate cancer. This condition leads to the spill-over of nanovesicles into the peripheral blood of prostate cancer patients, where the levels of tumor biomarkers expressed by exosomes, such as PSA-exosomes, may represent a novel, non-invasive clinical tool for the screening and early diagnosis of prostate cancer. (hide) EV-METRIC 25% (55th 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 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 Filtration Protein markers EV: CD81/ TSG101/ PSA non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma 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: speed (g) 110000 Wash: time (min) 60 Wash: speed (g) 110000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) particles per pellet ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins PSA Flow cytometry Type of Flow cytometry Cytoflex Hardware adaptation to ~100nm EV's The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Antibody details provided? Yes Detected EV-associated proteins CD81/ PSA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-250 EV concentration Yes Particle yield particles per pellet Particle analysis: flow cytometry Flow cytometer type Cytoflex Hardware adjustment The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Reported size (nm) <180 EV concentration Yes Particle yield particles per pellet

	EV220184	4/5	Homo sapiens	Blood plasma	(d)(U)C Filtration	Logozzi M	2017	25%
Study summary Full title Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients. All authors Logozzi M, Angelini DF, Iessi E, Mizzoni D, Di Raimo R, Federici C, Lugini L, Borsellino G, Gentilucci A, Pierella F, Marzio V, Sciarra A, Battistini L, Fais S Journal Cancer Lett Abstract Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis (show more...)Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis of prostate cancer (PCa). While neoplastic lesions of the prostate may cause aberrant levels of PSA in the blood, the quantitation of free or complexed PSA poorly discriminates cancer patients from those developing benign lesions, often leading to invasive and unnecessary surgical procedures. Microenvironmental acidity increases exosome release by cancer cells. In this study we evaluated whether acidity, a critical phenotype of malignancy, could influence exosome release and increase the PSA expression in nanovesicles released by PCa cells. To this aim, we exploited Nanoparticle Tracking Analysis (NTA), an immunocapture-based ELISA, and nanoscale flow-cytometry. The results show that microenvironmental acidity induces an increased release of nanovesicles expressing both PSA and the exosome marker CD81. In order to verify whether the changes induced by the local selective pressure of extracellular acidity may correspond to a clinical pathway we used the same approach to evaluate the levels of PSA-expressing exosomes in the plasma of PCa patients and controls, including subjects with benign prostatic hypertrophy (BPH). The results show that only PCa patients have high levels of nanovesicles expressing both CD81 and PSA. This study shows that tumor acidity exerts a selective pressure leading to the release of extracellular vesicles that express both PSA and exosome markers. A comparable scenario was shown in the plasma of prostate cancer patients as compared to both BPH and healthy controls. These results suggest that microenvironmental acidity may represent a key factor which determines qualitatively and quantitatively the release of extracellular vesicles by malignant tumors, including prostate cancer. This condition leads to the spill-over of nanovesicles into the peripheral blood of prostate cancer patients, where the levels of tumor biomarkers expressed by exosomes, such as PSA-exosomes, may represent a novel, non-invasive clinical tool for the screening and early diagnosis of prostate cancer. (hide) EV-METRIC 25% (55th 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 Prostate Cancer Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD81/ TSG101/ PSA non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma 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: speed (g) 110000 Wash: time (min) 60 Wash: speed (g) 110000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) particles per pellet ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins PSA Flow cytometry Type of Flow cytometry Cytoflex Hardware adaptation to ~100nm EV's The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Antibody details provided? Yes Detected EV-associated proteins CD81/ PSA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-250 EV concentration Yes Particle yield particles per pellet Particle analysis: flow cytometry Flow cytometer type Cytoflex Hardware adjustment The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Reported size (nm) <180 EV concentration Yes Particle yield particles per pellet

	EV220184	5/5	Homo sapiens	Blood plasma	(d)(U)C Filtration	Logozzi M	2017	25%
Study summary Full title Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients. All authors Logozzi M, Angelini DF, Iessi E, Mizzoni D, Di Raimo R, Federici C, Lugini L, Borsellino G, Gentilucci A, Pierella F, Marzio V, Sciarra A, Battistini L, Fais S Journal Cancer Lett Abstract Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis (show more...)Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis of prostate cancer (PCa). While neoplastic lesions of the prostate may cause aberrant levels of PSA in the blood, the quantitation of free or complexed PSA poorly discriminates cancer patients from those developing benign lesions, often leading to invasive and unnecessary surgical procedures. Microenvironmental acidity increases exosome release by cancer cells. In this study we evaluated whether acidity, a critical phenotype of malignancy, could influence exosome release and increase the PSA expression in nanovesicles released by PCa cells. To this aim, we exploited Nanoparticle Tracking Analysis (NTA), an immunocapture-based ELISA, and nanoscale flow-cytometry. The results show that microenvironmental acidity induces an increased release of nanovesicles expressing both PSA and the exosome marker CD81. In order to verify whether the changes induced by the local selective pressure of extracellular acidity may correspond to a clinical pathway we used the same approach to evaluate the levels of PSA-expressing exosomes in the plasma of PCa patients and controls, including subjects with benign prostatic hypertrophy (BPH). The results show that only PCa patients have high levels of nanovesicles expressing both CD81 and PSA. This study shows that tumor acidity exerts a selective pressure leading to the release of extracellular vesicles that express both PSA and exosome markers. A comparable scenario was shown in the plasma of prostate cancer patients as compared to both BPH and healthy controls. These results suggest that microenvironmental acidity may represent a key factor which determines qualitatively and quantitatively the release of extracellular vesicles by malignant tumors, including prostate cancer. This condition leads to the spill-over of nanovesicles into the peripheral blood of prostate cancer patients, where the levels of tumor biomarkers expressed by exosomes, such as PSA-exosomes, may represent a novel, non-invasive clinical tool for the screening and early diagnosis of prostate cancer. (hide) EV-METRIC 25% (55th 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 Benign prostatic hyperplasia 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 Filtration Protein markers EV: CD81/ TSG101/ PSA non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma 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: speed (g) 110000 Wash: time (min) 60 Wash: speed (g) 110000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) particles per pellet ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins PSA Flow cytometry Type of Flow cytometry Cytoflex Hardware adaptation to ~100nm EV's The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Antibody details provided? Yes Detected EV-associated proteins CD81/ PSA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-250 EV concentration Yes Particle yield particles per pellet Particle analysis: flow cytometry Flow cytometer type Cytoflex Hardware adjustment The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Reported size (nm) <180 EV concentration Yes Particle yield particles per pellet

	EV220171	3/4	Homo sapiens	Serum	ExoQuick	Xue M	2017	25%
Study summary Full title Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1. All authors Xue M, Chen W, Xiang A, Wang R, Chen H, Pan J, Pang H, An H, Wang X, Hou H, Li X Journal Mol Cancer Abstract To overcome the hostile hypoxic microenvironment of solid tumors, tumor cells secrete a large number (show more...)To overcome the hostile hypoxic microenvironment of solid tumors, tumor cells secrete a large number of non-coding RNA-containing exosomes that facilitate tumor development and metastasis. However, the precise mechanisms of tumor cell-derived exosomes during hypoxia are unknown. Here, we aim to clarify whether hypoxia affects tumor growth and progression by transferring long non-coding RNA-urothelial cancer-associated 1 (lncRNA-UCA1) enriched exosomes secreted from bladder cancer cells. (hide) EV-METRIC 25% (67th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Protein markers EV: CD63/ HSP70/ HSP90/ TSG101 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63/ HSP70/ HSP90/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 50200

	EV220171	4/4	Homo sapiens	Serum	ExoQuick	Xue M	2017	25%
Study summary Full title Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1. All authors Xue M, Chen W, Xiang A, Wang R, Chen H, Pan J, Pang H, An H, Wang X, Hou H, Li X Journal Mol Cancer Abstract To overcome the hostile hypoxic microenvironment of solid tumors, tumor cells secrete a large number (show more...)To overcome the hostile hypoxic microenvironment of solid tumors, tumor cells secrete a large number of non-coding RNA-containing exosomes that facilitate tumor development and metastasis. However, the precise mechanisms of tumor cell-derived exosomes during hypoxia are unknown. Here, we aim to clarify whether hypoxia affects tumor growth and progression by transferring long non-coding RNA-urothelial cancer-associated 1 (lncRNA-UCA1) enriched exosomes secreted from bladder cancer cells. (hide) EV-METRIC 25% (67th 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 Bladder Cancer Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture ExoQuick Protein markers EV: CD63/ HSP70/ HSP90/ TSG101 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD63/ HSP70/ HSP90/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 50200

	EV210319	1/7	Homo sapiens	MDAMB231	(d)(U)C ExoQuick	Di Modica M	2017	25%
Study summary Full title Breast cancer-secreted miR-939 downregulates VE-cadherin and destroys the barrier function of endothelial monolayers. All authors Di Modica M, Regondi V, Sandri M, Iorio MV, Zanetti A, Tagliabue E, Casalini P, Triulzi T Journal Cancer Lett Abstract Exosomes-secreted microRNAs play an important role in metastatic spread. During this process breast (show more...)Exosomes-secreted microRNAs play an important role in metastatic spread. During this process breast cancer cells acquire the ability to transmigrate through blood vessels by inducing changes in the endothelial barrier. We focused on miR-939 that is predicted to target VE-cadherin, a component of adherens junction involved in vessel permeability. By in silico analysis miR-939 was found highly expressed in the basal-like tumor subtypes and in our cohort of 63 triple-negative breast cancers (TNBCs) its expression significantly interacted with lymph node status in predicting disease-free survival probability. We demonstrated, in vitro, that miR-939 directly targets VE-cadherin leading to an increase in HUVECs monolayer permeability. MDA-MB-231 cells transfected with a miR-939 mimic, released miR-939 in exosomes that, once internalized in endothelial cells, favored trans-endothelial migration of MDA-MB-231-GFP cells by the disruption of the endothelial barrier. Notably, when up taken in endothelial cells exosomes caused VE-cadherin down-regulation specifically through miR-939 as we demonstrated by inhibiting miR-939 expression in exosomes-releasing TNBC cells. Together, our data indentify an extracellular pro-tumorigenic role for tumor-derived, exosome-associated miR-939 that can explain its association with worse prognosis in TNBCs. (hide) EV-METRIC 25% (64th 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 ExoQuick Protein markers EV: CD63 non-EV: tubulin Proteomics no Show all info Study aim Function/ Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63 Detected contaminants tubulin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210195	1/7	Homo sapiens	T24	(d)(U)C Total Exosome Isolation	Baumgart, Sophie	2017	25%
Study summary Full title Exosomes of invasive urothelial carcinoma cells are characterized by a specific miRNA expression signature All authors Sophie Baumgart, Sebastian Hölters, Carsten-Henning Ohlmann, Rainer Bohle, Michael Stöckle, Marie Stampe Ostenfeld, Lars Dyrskjøt, Kerstin Junker, Joana Heinzelmann Journal Oncotarget Abstract Muscle-invasive bladder cancer (MIBC) represents a highly aggressive tumor type compared to non-musc (show more...)Muscle-invasive bladder cancer (MIBC) represents a highly aggressive tumor type compared to non-muscle-invasive tumors. MIBC is characterized by specific molecular alterations, which may also modulate extracellular tumorigenic effects. Tumor-associated exosomes, especially exosomal miRNAs, are important regulators in the interaction between tumor cells and tumor microenvironment by affecting tumor-promoting processes in target cells. It is important to analyze whether their exosomal patterns also reflect the specific molecular characteristics of MIBC. The aim of this study was to compare the miRNA expression in secreted exosomes from urinary bladder cancer cells (UBC) with different degrees of invasiveness. By electron microscopy, nanotracking analysis and western blot we proofed a high quality of isolated exosomes. Microarray analysis identified an invasion-associated signature of 15 miRNAs, which is significantly altered in exosomes of invasive UBC compared to non-invasive counterparts. Therefrom, 9 miRNAs are consistent differently expressed in both, invasive cells and their secreted exosomes. The remaining 6 exosome-specific miRNAs are only deregulated in exosomes but not in their parental cells. MiRNA alterations were verified by qPCR in cell culture and urinary exosomes. In conclusion, we showed that exosomes from invasive UBC cells are characterized by a specific miRNA signature. Further analyses have to clarify the functional relevance of exosomal miRNAs secreted by invasive bladder cancer cells for modification of the tumor microenvironment and their putative role as molecular markers in liquid biopsies. (hide) EV-METRIC 25% (64th 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 (d)(U)C Commercial method Protein markers EV: CD81/ Synthenin non-EV: Calreticulin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells T24 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Synthenin/ CD81 Not detected contaminants Calreticulin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type Other;RNase ONE Ribonuclease (Promega) RNAse concentration 10 units Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 75 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 57

	EV210193	6/6	Homo sapiens	Urine	Hydrostatic filtration dialysis	Xu, Yong	2017	25%
Study summary Full title MiR-145 detection in urinary extracellular vesicles increase diagnostic efficiency of prostate cancer based on hydrostatic filtration dialysis method All authors Yong Xu, Sihua Qin, Taixue An, Yueting Tang, Yiyao Huang, Lei Zheng Journal Prostate Abstract Background: Extracellular vesicles (EVs) can be detected in body fluids and may serve as disease bio (show more...)Background: Extracellular vesicles (EVs) can be detected in body fluids and may serve as disease biomarkers. Increasing evidence suggests that circulating miRNAs in serum and urine may be potential non-invasive biomarkers for prostate cancer (PCa). In the present study, we aimed to investigate whether hydrostatic filtration dialysis (HFD) is suitable for urinary EVs (UEVs) isolation and whether such reported PCa-related miRNAs can be detected in UEVs as PCa biomarkers. Methods: To analyze EVs miRNAs, we searched for an easy and economic method to enrich EVs from urine samples. We compared the efficiency of HFD method and conventional ultracentrifugation (UC) in isolating UEVs. Subsequently, UEVs were isolated from patients with PCa, patients with benign prostate hyperplasia (BPH) and healthy individuals. Differential expression of four PCa-related miRNAs (miR-572, miR-1290, miR-141, and miR-145) were measured in UEVs and paired serum EVs using SYBR Green-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: The overall performance of HFD was similar to UC. In miRNA yield, both HFD and UC can meet the needs of further analysis. The level of miR-145 in UEVs was significantly increased in patients with PCa compared with the patients with BPH (P = 0.018). In addition, significant increase was observed in miR-145 levels when patients with Gleason score ≥8 tumors compared with Gleason score ≤7 (P = 0.020). Receiver-operating characteristic curve (ROC) revealed that miR-145 in UEVs combined with serum PSA could differentiate PCa from BPH better than PSA alone (AUC 0.863 and AUC 0.805, respectively). In serum EVs, four miRNAs were significantly higher in patients with PCa than with BPH. Conclusion: HFD is appropriate for UEVs isolation and miRNA analysis when compared with conventional UC. miR-145 in UEVs is upregulated from PCa patients compared BPH patients and healthy controls. We suggest the potential use of UEVs miR-145 as a biomarker of PCa. (hide) EV-METRIC 25% (56th 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 Prostate cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Hydrostatic filtration dialysis Protein markers EV: Alix/ TSG101/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods/Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method Other Name other separation method Hydrostatic filtration dialysis Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ CD9/ CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EM EM-type Transmission-EM Image type Close-up

	EV200137	1/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (55th 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 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 Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200137	2/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated annexin AV-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (55th 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 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 Streptavidin bead precipitation of biotinylated annexin AV-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Streptavidin bead precipitation of biotinylated annexin AV-bound EV Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200137	3/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (55th 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 Pre-eclampsia 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 Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200137	4/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated annexin AV-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (55th 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 Pre-eclampsia 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 Streptavidin bead precipitation of biotinylated annexin AV-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Streptavidin bead precipitation of biotinylated annexin AV-bound EV Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV170005	3/4	Homo sapiens	SK-MEL103	(d)(U)C	Suárez H	2017	25%
Study summary Full title A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles. All authors Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, Cabañas C, Borràs FE, Yáñez-Mó M Journal Sci Rep Abstract Most experimental approaches commonly employed for the characterization and quantitation of EVs are (show more...)Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories. (hide) EV-METRIC 25% (64th 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 Adj. k-factor 264.9 (pelleting) / 264.9 (washing) Protein markers EV: CD59/ CD9 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-MEL103 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 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 AH-627 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 264.9 Wash: time (min) 120 Wash: Rotor Type AH-627 Wash: speed (g) 100000 Wash: adjusted k-factor 264.9 Characterization: Protein analysis Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median

	EV220184	1/5	Homo sapiens	LNCap	(d)(U)C Filtration	Logozzi M	2017	23%
Study summary Full title Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients. All authors Logozzi M, Angelini DF, Iessi E, Mizzoni D, Di Raimo R, Federici C, Lugini L, Borsellino G, Gentilucci A, Pierella F, Marzio V, Sciarra A, Battistini L, Fais S Journal Cancer Lett Abstract Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis (show more...)Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis of prostate cancer (PCa). While neoplastic lesions of the prostate may cause aberrant levels of PSA in the blood, the quantitation of free or complexed PSA poorly discriminates cancer patients from those developing benign lesions, often leading to invasive and unnecessary surgical procedures. Microenvironmental acidity increases exosome release by cancer cells. In this study we evaluated whether acidity, a critical phenotype of malignancy, could influence exosome release and increase the PSA expression in nanovesicles released by PCa cells. To this aim, we exploited Nanoparticle Tracking Analysis (NTA), an immunocapture-based ELISA, and nanoscale flow-cytometry. The results show that microenvironmental acidity induces an increased release of nanovesicles expressing both PSA and the exosome marker CD81. In order to verify whether the changes induced by the local selective pressure of extracellular acidity may correspond to a clinical pathway we used the same approach to evaluate the levels of PSA-expressing exosomes in the plasma of PCa patients and controls, including subjects with benign prostatic hypertrophy (BPH). The results show that only PCa patients have high levels of nanovesicles expressing both CD81 and PSA. This study shows that tumor acidity exerts a selective pressure leading to the release of extracellular vesicles that express both PSA and exosome markers. A comparable scenario was shown in the plasma of prostate cancer patients as compared to both BPH and healthy controls. These results suggest that microenvironmental acidity may represent a key factor which determines qualitatively and quantitatively the release of extracellular vesicles by malignant tumors, including prostate cancer. This condition leads to the spill-over of nanovesicles into the peripheral blood of prostate cancer patients, where the levels of tumor biomarkers expressed by exosomes, such as PSA-exosomes, may represent a novel, non-invasive clinical tool for the screening and early diagnosis of prostate cancer. (hide) EV-METRIC 23% (62nd 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 Filtration Protein markers EV: CD81/ TSG101/ PSA non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCap EV-harvesting Medium EV-depleted medium Preparation of EDS 0.45µm and 0.2µm filtration, 110,000g centrifugation, duration NS 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: speed (g) 110000 Wash: time (min) 60 Wash: speed (g) 110000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) particles per pellet Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101/ PSA ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins PSA Flow cytometry Type of Flow cytometry Cytoflex Hardware adaptation to ~100nm EV's The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Antibody details provided? Yes Detected EV-associated proteins CD81/ PSA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-250 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Cytoflex Hardware adjustment The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Reported size (nm) <180 EV concentration Yes Particle yield particles per pellet: 5.00e+3

	EV220184	2/5	Homo sapiens	LNCap	(d)(U)C Filtration	Logozzi M	2017	23%
Study summary Full title Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients. All authors Logozzi M, Angelini DF, Iessi E, Mizzoni D, Di Raimo R, Federici C, Lugini L, Borsellino G, Gentilucci A, Pierella F, Marzio V, Sciarra A, Battistini L, Fais S Journal Cancer Lett Abstract Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis (show more...)Prostate specific antigen (PSA) test is the most common, clinically validated test for the diagnosis of prostate cancer (PCa). While neoplastic lesions of the prostate may cause aberrant levels of PSA in the blood, the quantitation of free or complexed PSA poorly discriminates cancer patients from those developing benign lesions, often leading to invasive and unnecessary surgical procedures. Microenvironmental acidity increases exosome release by cancer cells. In this study we evaluated whether acidity, a critical phenotype of malignancy, could influence exosome release and increase the PSA expression in nanovesicles released by PCa cells. To this aim, we exploited Nanoparticle Tracking Analysis (NTA), an immunocapture-based ELISA, and nanoscale flow-cytometry. The results show that microenvironmental acidity induces an increased release of nanovesicles expressing both PSA and the exosome marker CD81. In order to verify whether the changes induced by the local selective pressure of extracellular acidity may correspond to a clinical pathway we used the same approach to evaluate the levels of PSA-expressing exosomes in the plasma of PCa patients and controls, including subjects with benign prostatic hypertrophy (BPH). The results show that only PCa patients have high levels of nanovesicles expressing both CD81 and PSA. This study shows that tumor acidity exerts a selective pressure leading to the release of extracellular vesicles that express both PSA and exosome markers. A comparable scenario was shown in the plasma of prostate cancer patients as compared to both BPH and healthy controls. These results suggest that microenvironmental acidity may represent a key factor which determines qualitatively and quantitatively the release of extracellular vesicles by malignant tumors, including prostate cancer. This condition leads to the spill-over of nanovesicles into the peripheral blood of prostate cancer patients, where the levels of tumor biomarkers expressed by exosomes, such as PSA-exosomes, may represent a novel, non-invasive clinical tool for the screening and early diagnosis of prostate cancer. (hide) EV-METRIC 23% (62nd 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 pH 6.5 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 Filtration Protein markers EV: CD81/ TSG101/ PSA non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCap EV-harvesting Medium EV-depleted medium Preparation of EDS 0.45µm and 0.2µm filtration, 110,000g centrifugation, duration NS 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: speed (g) 110000 Wash: time (min) 60 Wash: speed (g) 110000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) particles per pellet Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101/ PSA ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins PSA Flow cytometry Type of Flow cytometry Cytoflex Hardware adaptation to ~100nm EV's The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Antibody details provided? Yes Detected EV-associated proteins CD81/ PSA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 110-180 EV concentration Yes Particle yield particles per pellet Particle analysis: flow cytometry Flow cytometer type Cytoflex Hardware adjustment The Cytoflex flow cytometer is equipped with custom fluidics and with the possibility of using the Violet (405 nm) Side Scatter (VSSC) as a trigger parameter to better resolve nanoparticles with the VSSC parameter. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes ranging from 110 nm to 1300 nm. Calibration bead size 110-1300 Reported size (nm) 180-590 EV concentration Yes Particle yield particles per pellet: 2.00e+4

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