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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190108	3/6	Homo sapiens	Tumour tissue	DG (d)(U)C	Crescitelli R	2019	100%
Study summary Full title Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. All authors Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög JL, Johansson I, Fuchs J, Thorsell A, Gho YS, Olofsson Bagge R, Lötvall J. Journal J Extracell Vesicles Abstract The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lin (show more...)The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery. (hide) EV-METRIC 100% (66th 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 Tumour tissue Sample origin Metastatic melanoma Focus vesicles Other / Large low density extracellular vesicles (Large LD EVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Mitofilin/ CD63/ CD81/ ADAM10/ Flotillin1/ CD9 non-EV: CD41a Proteomics yes EV density (g/ml) 1.111-1.121 Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Tumour tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 20% Highest density fraction 45-57,3% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.18-1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 186000 Duration (min) 960 Fraction volume (mL) 1-1.3 Fraction processing None Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Mitofilin/ ADAM10/ CD81 Proteomics database Yes: Other 1 ExoView Detected EV-associated proteins CD81/ CD9/ CD63 Not detected contaminants CD41a Characterization: RNA analysis RNA analysis Type 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 EV concentration Yes Particle yield as number/g tumour tissue;Yes, other: 2.17E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 124

	EV190108	4/6	Homo sapiens	Tumour tissue	DG (d)(U)C	Crescitelli R	2019	100%
Study summary Full title Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. All authors Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög JL, Johansson I, Fuchs J, Thorsell A, Gho YS, Olofsson Bagge R, Lötvall J. Journal J Extracell Vesicles Abstract The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lin (show more...)The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery. (hide) EV-METRIC 100% (66th 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 Tumour tissue Sample origin Metastatic melanoma Focus vesicles Other / Large high density extracellular vesicles (Large HD EVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Mitofilin/ CD63/ CD81/ non/ ADAM10/ Flotillin1/ CD9 non-EV: CD41a Proteomics yes EV density (g/ml) 1.163-1.189 Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Tumour tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 20 Highest density fraction 45-57.3 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0,18-1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 186000 Duration (min) 960 Fraction volume (mL) 0.5-1.2 Fraction processing None Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins non Not detected EV-associated proteins CD81/ Flotillin1/ Mitofilin/ ADAM10 Proteomics database Yes: Other 1 ExoView Detected EV-associated proteins CD81/ CD9/ CD63 Not detected contaminants CD41a Characterization: RNA analysis RNA analysis Type 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 EV concentration Yes Particle yield as number/g tumour tissue;Yes, other: 3.21E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 39

	EV190108	5/6	Homo sapiens	Tumour tissue	DG (d)(U)C	Crescitelli R	2019	100%
Study summary Full title Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. All authors Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög JL, Johansson I, Fuchs J, Thorsell A, Gho YS, Olofsson Bagge R, Lötvall J. Journal J Extracell Vesicles Abstract The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lin (show more...)The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery. (hide) EV-METRIC 100% (66th 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 Tumour tissue Sample origin Metastatic melanoma Focus vesicles Other / Small low density extracellular vesicles (Small LD EVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Mitofilin/ CD63/ CD81/ ADAM10/ Flotillin1/ CD9 non-EV: CD41a Proteomics yes EV density (g/ml) 1.111-1.121 Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Tumour tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 11800 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 20 Highest density fraction 45-57.3 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.18-1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 186000 Duration (min) 960 Fraction volume (mL) 0.8-1.2 Fraction processing None Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ ADAM10/ CD81 Not detected EV-associated proteins Mitofilin Proteomics database Yes: Other 1 ExoView Detected EV-associated proteins CD81/ CD9/ CD63 Not detected contaminants CD41a Characterization: RNA analysis RNA analysis Type 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 EV concentration Yes Particle yield as number/g tumour tissue;Yes, other: 1.11E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 67

	EV190108	6/6	Homo sapiens	Tumour tissue	DG (d)(U)C	Crescitelli R	2019	100%
Study summary Full title Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. All authors Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög JL, Johansson I, Fuchs J, Thorsell A, Gho YS, Olofsson Bagge R, Lötvall J. Journal J Extracell Vesicles Abstract The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lin (show more...)The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery. (hide) EV-METRIC 100% (66th 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 Tumour tissue Sample origin Metastatic melanoma Focus vesicles Other / Small high density extracellular vesicles (Small HD EVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: Mitofilin/ CD63/ CD81/ non/ ADAM10/ Flotillin1/ CD9 non-EV: CD41a Proteomics yes EV density (g/ml) 1.163-1.189 Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Tumour tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118000 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 20 Highest density fraction 45-57.3 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.18-1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 186000 Duration (min) 960 Fraction volume (mL) 0.5-1.2 Fraction processing None Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins non Not detected EV-associated proteins CD81/ Flotillin1/ ADAM10/ Mitofilin Proteomics database Yes: Other 1 ExoView Detected EV-associated proteins CD81/ CD9/ CD63 Not detected contaminants CD41a Characterization: RNA analysis RNA analysis Type 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 EV concentration Yes Particle yield as number/g tumour tissue;Yes, other: 4.77E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30

	EV190045	1/4	Homo sapiens	HMC-1	DG (d)(U)C	Lázaro-Ibáñez E	2019	100%
Study summary Full title DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology. All authors Lázaro-Ibáñez E, Lässer C, Shelke GV, Crescitelli R, Jang SC, Cvjetkovic A, García-Rodríguez A, Lötvall J. Journal J Extracell Vesicles Abstract Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cel (show more...)Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell's phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low- and high-density sEV subsets from a human mast cell line (HMC-1) and an erythroleukemic cell line (TF-1) were separated using high-resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV-associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high-density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole-genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ BrdU/ beta-actin/ CD9 non-EV: Calnexin Proteomics yes EV density (g/ml) 1.111-1.133 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118500 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 180000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 94 Pelleting: duration (min) 210 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118500 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ TSG101/ Alix/ CD81 Not detected EV-associated proteins beta-actin Not detected contaminants Calnexin ELISA Detected EV-associated proteins BrdU/ CD9 Flow cytometry specific beads Detected EV-associated proteins CD63 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190045	2/4	Homo sapiens	HMC-1	DG (d)(U)C	Lázaro-Ibáñez E	2019	100%
Study summary Full title DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology. All authors Lázaro-Ibáñez E, Lässer C, Shelke GV, Crescitelli R, Jang SC, Cvjetkovic A, García-Rodríguez A, Lötvall J. Journal J Extracell Vesicles Abstract Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cel (show more...)Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell's phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low- and high-density sEV subsets from a human mast cell line (HMC-1) and an erythroleukemic cell line (TF-1) were separated using high-resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV-associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high-density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole-genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: TSG101/ Histone H3/ CD63/ CD81/ Alix/ Flotillin1/ Histone H2A/ beta-actin/ CD9 non-EV: Calnexin Proteomics yes EV density (g/ml) 1.144-1.176 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HMC-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118500 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 180000 Duration (min) 960 Fraction volume (mL) 4 Fraction processing Centrifugation Pelleting: volume per fraction 94 Pelleting: duration (min) 210 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118500 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD9/ CD63/ beta-actin/ TSG101/ CD81/ Histone H2A/ Histone H3 Detected contaminants Calnexin ELISA Detected EV-associated proteins CD9 Flow cytometry specific beads Detected EV-associated proteins CD63 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-300 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190045	3/4	Homo sapiens	TF-1	DG (d)(U)C	Lázaro-Ibáñez E	2019	100%
Study summary Full title DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology. All authors Lázaro-Ibáñez E, Lässer C, Shelke GV, Crescitelli R, Jang SC, Cvjetkovic A, García-Rodríguez A, Lötvall J. Journal J Extracell Vesicles Abstract Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cel (show more...)Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell's phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low- and high-density sEV subsets from a human mast cell line (HMC-1) and an erythroleukemic cell line (TF-1) were separated using high-resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV-associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high-density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole-genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD9 non-EV: Calnexin Proteomics no EV density (g/ml) 1.103-1.137 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells TF-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118500 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 180000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 94 Pelleting: duration (min) 210 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118500 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD63/ TSG101/ CD81 Not detected EV-associated proteins CD9 Not detected contaminants Calnexin Flow cytometry specific beads Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190045	4/4	Homo sapiens	TF-1	DG (d)(U)C	Lázaro-Ibáñez E	2019	100%
Study summary Full title DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology. All authors Lázaro-Ibáñez E, Lässer C, Shelke GV, Crescitelli R, Jang SC, Cvjetkovic A, García-Rodríguez A, Lötvall J. Journal J Extracell Vesicles Abstract Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cel (show more...)Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell's phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low- and high-density sEV subsets from a human mast cell line (HMC-1) and an erythroleukemic cell line (TF-1) were separated using high-resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV-associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high-density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole-genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: TSG101/ Histone H3/ CD63/ CD81/ Alix/ Flotillin1/ Histone H3/ Histone H2A/ beta-actin/ CD9 non-EV: Calnexin Proteomics no EV density (g/ml) 1.148-1.192 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells TF-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118500 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 180000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 94 Pelleting: duration (min) 210 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 1185000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ beta-actin/ CD9/ CD63/ TSG101/ CD81/ Histone H2A/ Histone H3 Not detected EV-associated proteins Alix Not detected contaminants Calnexin Flow cytometry specific beads Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-300 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	3/8	Homo sapiens	MKN45	DG (d)(U)C Filtration	Freitas D	2019	100%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Protein markers EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1 non-EV: Albumin Proteomics yes EV density (g/ml) 1.05-1.10 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 5.5 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Ultrafiltration Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ Syntenin-1 Not detected EV-associated proteins CD81 Detected contaminants Albumin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131.65 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	7/8	Homo sapiens	MKN45	DG (d)(U)C Filtration	Freitas D	2019	100%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin COSMC KO Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Protein markers EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1 non-EV: Albumin Proteomics yes EV density (g/ml) 1.05-1.10 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 5.5 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Ultrafiltration Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Alix/ Syntenin-1/ CD9/ CD63/ HSP70/ CD81 Detected contaminants Albumin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 121.05 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190011	4/5	Mus musculus	Tissue	DG (d)(U)C	Cianciaruso C	2019	100%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 100% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Tissue Sample origin MC38 tumor Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: / TSG101/ CD63/ TBXAS1/ MRC1/ CD81/ COX1/ GAPDH/ CD68/ Alix/ actin-beta/ HER2/ CD9/ CD11b non-EV: Calnexin/ Gp96 Proteomics yes EV density (g/ml) 1.14 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 17% Highest density fraction 78% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 35 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 134000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101/ CD81/ GAPDH/ MRC1/ CD68/ actin-beta/ HER2/ TBXAS1/ COX1 Detected contaminants Calnexin/ Gp96 Flow cytometry specific beads Detected EV-associated proteins CD11b/ CD9 Flow cytometry Type of Flow cytometry Attune NxT apparatus Hardware adaptation to ~100nm EV's Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep Detected EV-associated proteins Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 190 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190011	5/5	Mus musculus	Tissue	DG (d)(U)C	Cianciaruso C	2019	100%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 100% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Tissue Sample origin E0771 tumor Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: MRC1/ COX1/ CD9/ TBXAS1 non-EV: Gp96 Proteomics yes EV density (g/ml) 1.14 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Density gradient Type Continuous Lowest density fraction 17% Highest density fraction 77% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 35 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 134000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ MRC1/ COX1/ TBXAS1 Detected contaminants Gp96 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190007	1/5	Homo sapiens	Urine	DG (d)(U)C UF	Mussack V	2019	100%
Study summary Full title Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing-Enabling robust and non-invasive biomarker research. All authors Mussack V, Wittmann G, Pfaffl MW. Journal Biomol Detect Quanti Abstract Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication th (show more...)Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 DG (d)(U)C UF Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Syntenin/ EPCAM/ HSP70/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein Proteomics no EV density (g/ml) 1.18-1.24 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 SW 60 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 9.75 Sample volume (mL) 0.75 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.9 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: duration (min) 60 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ Syntenin/ TSG101/ Alix Not detected EV-associated proteins HSP70/ CD81/ EPCAM/ CD63 Detected contaminants Tamm-Horsfall protein Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180011	1/1	Homo sapiens	HEK293	DG (d)(U)C Filtration	Kathrin Gärtner	2019	100%
Study summary Full title Engineering extracellular vesicles as novel treatment options: exploiting herpesviral immunity in CLL All authors Kathrin Gärtner, Manja Luckner, Gerhard Wanner, Reinhard Zeidler Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are important mediators of cell–cell communication. Intriguingly, EVs (show more...)Extracellular vesicles (EVs) are important mediators of cell–cell communication. Intriguingly, EVs can be engineered and thus exploited for the targeted transfer of functional proteins of interest. Thus, engineered EVs may constitute attractive tools for the development of novel therapeutic interventions, like cancer immunotherapies, vaccinations or targeted drug delivery. Here, we describe a novel experimental immunotherapeutic approach for the adjuvant treatment of chronic lymphocytic leukaemia (CLL) based on engineered EVs carrying gp350, the major glycoprotein of Epstein–Barr virus (EBV), CD40L, a central immune accessory molecule and pp65, an immunodominant antigen of the human cytomegalovirus (CMV). We show that these engineered EVs specifically interact with malignant B cells from CLL patients and render these cells immunogenic to allogeneic and autologous EBV- and CMV-specific CD4+ and CD8+ T cells. Collectively, co-opting engineered EVs to re-target the strong herpesviral immunity in CLL patients to malignant cells constitutes an attractive strategy for the adjuvant treatment of a still incurable disease. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Overexpressing gp350, CD40L and/or pp65 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Filtration Adj. k-factor 253.9 (pelleting) Protein markers EV: Alix/ TSG101/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Density gradient Type Discontinuous Number of initial discontinuous layers 2 Lowest density fraction 0.3 Highest density fraction 0.44 Sample volume (mL) 0.5 Orientation Bottom-up (sample migrates upwards) Rotor type SW 60 Ti Speed (g) 160000 Duration (min) 960 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 30 Pelleting: duration (min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins Alix, CD63, TSG101 Not detected contaminants Calnexin Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes EM EM-type Transmission-EM/ Immune-EM EM protein CD63 Image type Close-up, Wide-field

	EV190040	1/12	Homo sapiens	HEK293T	DG UF	Geeurickx E	2019	88%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 88% (98th 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 gag-EGFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: TSG101/ CD81/ Alix/ p24/ CD9/ syntenin-1 non-EV: Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium Cell viability (%) 96 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ syntenin-1/ TSG101/ Alix/ CD81 ELISA Detected EV-associated proteins p24 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 108.6 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type High resolution flow cytometry Hardware adjustment 200 mW 488 nm laser (Sapphire; Coherent, Santa Clara, CA, USA) and a large-bore nozzle (140 m) were used, sheath pressure was permanently monitored and kept within 4.89 to 5.02 psi, and the sample pressure was set at 4.29 psi, to assure an identical diameter of the core in the jet stream. Forward scattered light was measured with a collection angle of 1525 (reduced wide-angle forward scatter [rw-FSC]). Calibration bead size 0.102 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field

	EV190040	2/12	Homo sapiens	HEK293T	DG UF	Geeurickx E	2019	88%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 88% (98th 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 gag-EGFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: TSG101/ CD81/ Alix/ p24/ CD9/ syntenin-1 non-EV: Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium Cell viability (%) 96 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ syntenin-1/ TSG101/ Alix/ CD81 ELISA Detected EV-associated proteins p24 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 108.6 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type High resolution flow cytometry Hardware adjustment 200 mW 488 nm laser (Sapphire; Coherent, Santa Clara, CA, USA) and a large-bore nozzle (140 m) were used, sheath pressure was permanently monitored and kept within 4.89 to 5.02 psi, and the sample pressure was set at 4.29 psi, to assure an identical diameter of the core in the jet stream. Forward scattered light was measured with a collection angle of 1525 (reduced wide-angle forward scatter [rw-FSC]). Calibration bead size 0.102 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD63 Image type Close-up, Wide-field

	EV190040	5/12	Homo sapiens	HEK293T	DG UF	Geeurickx E	2019	88%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 88% (98th 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 mock Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: TSG101/ CD81/ Alix/ Flotillin1/ CD9/ syntenin-1 non-EV: Proteomics yes EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium Cell viability (%) 96 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ Alix/ syntenin-1/ CD9/ TSG101/ CD81 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190040	6/12	Homo sapiens	HEK293T	DG UF	Geeurickx E	2019	88%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 88% (98th 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 mock Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: TSG101/ CD81/ Alix/ Flotillin1/ CD9/ syntenin-1 non-EV: Proteomics yes EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium Cell viability (%) 96 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ Alix/ syntenin-1/ CD9/ TSG101/ CD81 Proteomics database Yes: Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190040	9/12	Homo sapiens	Blood plasma	DG UF	Geeurickx E	2019	88%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 88% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: TSG101/ CD81/ Alix/ Flotillin1/ CD9/ syntenin-1 non-EV: Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Sizeexclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ syntenin-1/ TSG101/ CD9/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 105 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190040	10/12	Homo sapiens	Blood plasma	DG UF	Geeurickx E	2019	88%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 88% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Breast cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: TSG101/ CD81/ Alix/ Flotillin1/ CD9/ syntenin-1 non-EV: Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ syntenin-1/ TSG101/ CD9/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190040	11/12	Homo sapiens	Urine	DG UF	Geeurickx E	2019	88%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 88% (98th 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 DG UF Protein markers EV: TSG101/ CD81/ Alix/ Flotillin1/ CD9/ syntenin-1 non-EV: Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD9/ syntenin-1/ TSG101/ Alix/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 105 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190040	12/12	Mus musculus	4T1	DG UF	Geeurickx E	2019	88%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 88% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: TSG101/ CD81/ Alix/ Flotillin1/ CD9/ syntenin-1 non-EV: Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD9/ syntenin-1/ TSG101/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 103 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190039	1/3	Homo sapiens	MCF-7	DG Filtration UF	Everaert, Celine	2019	88%
Study summary Full title Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles All authors Celine Everaert, Hetty Helsmoortel, Anneleen Decock, Eva Hulstaert, Ruben Van Paemel, Kimberly Verniers, Justine Nuytens 1 2 , Jasper Anckaert 1 2 , Nele Nijs, Joeri Tulkens, Bert Dhondt, An Hendrix, Pieter Mestdagh, Jo Vandesompele Journal Scientific Reports Abstract RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human bioflui (show more...)RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles. (hide) EV-METRIC 88% (98th 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 GFP-Rab27b transfected Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG Filtration UF Protein markers EV: TSG101/ Alix/ CD9 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.1 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-7 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1091 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 15 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EM EM-type Transmission-EM Image type Wide-field

	EV190039	3/3	Homo sapiens	Urine	DG (d)(U)C UF	Everaert, Celine	2019	88%
Study summary Full title Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles All authors Celine Everaert, Hetty Helsmoortel, Anneleen Decock, Eva Hulstaert, Ruben Van Paemel, Kimberly Verniers, Justine Nuytens 1 2 , Jasper Anckaert 1 2 , Nele Nijs, Joeri Tulkens, Bert Dhondt, An Hendrix, Pieter Mestdagh, Jo Vandesompele Journal Scientific Reports Abstract RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human bioflui (show more...)RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles. (hide) EV-METRIC 88% (98th 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 DG (d)(U)C UF Protein markers EV: Alix/ TSG101/ CD9 non-EV: Tamm-Horsfall protein Proteomics no EV density (g/ml) 1.1 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1091 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101 Detected contaminants Tamm-Horsfall protein Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EM EM-type Transmission-EM Image type Wide-field

	EV180002	2/2	Canis familiaris	Mesenchymal stromal cells of placental origin	DG (d)(U)C	Thane KE	2019	88%
Study summary Full title Improved methods for fluorescent labeling and detection of single extracellular vesicles using nanoparticle tracking analysis. All authors Thane KE, Davis AM, Hoffman AM. Journal Sci Rep Abstract Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve (show more...)Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration. (hide) EV-METRIC 88% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 122.2 (pelleting) Protein markers EV: TSG101/ CD81/ CD9 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Canis familiaris Sample Type Cell culture supernatant EV-producing cells Mesenchymal stromal cells of placental origin 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) 120 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 122.2 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.1 Highest density fraction 0.4 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 120 Fraction volume (mL) 0.625 Fraction processing Ultrafiltration Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9, TSG101 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes EM EM-type Immune-EM/ Atomic force-EM EM protein CD9 Image type Close-up, Wide-field

	EV190108	1/6	Homo sapiens	Tumour tissue	(d)(U)C	Crescitelli R	2019	78%
Study summary Full title Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. All authors Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög JL, Johansson I, Fuchs J, Thorsell A, Gho YS, Olofsson Bagge R, Lötvall J. Journal J Extracell Vesicles Abstract The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lin (show more...)The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery. (hide) EV-METRIC 78% (16th 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 Tumour tissue Sample origin Metastatic melanoma Focus vesicles Other / Large extracellular vesicles (Large EVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Mitofilin/ CD63/ CD81/ ADAM10/ Flotillin1/ CD9 non-EV: Calnexin/ CD41a Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Tumour tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Characterization: Protein analysis Protein Concentration Method Other;BCA;Qubit Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ Mitofilin/ ADAM10/ CD81 Detected contaminants Calnexin ELISA Detected EV-associated proteins CD63/ CD81/ CD9 Proteomics database Yes: Other 1 ExoView Detected EV-associated proteins CD9/ CD63/ CD81 Detected contaminants CD41a Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 125.7 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 142

	EV190108	2/6	Homo sapiens	Tumour tissue	(d)(U)C	Crescitelli R	2019	78%
Study summary Full title Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. All authors Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög JL, Johansson I, Fuchs J, Thorsell A, Gho YS, Olofsson Bagge R, Lötvall J. Journal J Extracell Vesicles Abstract The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lin (show more...)The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery. (hide) EV-METRIC 78% (16th 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 Tumour tissue Sample origin Metastatic melanoma Focus vesicles Other / Small extracellular vesicles (Small EVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Mitofilin/ CD63/ CD81/ ADAM10/ Flotillin1/ CD9 non-EV: Calnexin/ CD41a Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Tumour tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method Other;BCA;Qubit Western Blot Detected EV-associated proteins Flotillin1/ CD9/ CD63/ ADAM10/ CD81 Not detected EV-associated proteins Mitofilin Detected contaminants Calnexin ELISA Detected EV-associated proteins CD63/ CD81/ CD9 Proteomics database Yes: Other 1 ExoView Detected EV-associated proteins CD81/ CD9/ CD63 Detected contaminants CD41a Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 122 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 75

	EV190104	1/2	Mus musculus	32D	(d)(U)C	Swatler J	2019	78%
Study summary Full title Chronic myeloid leukemia-derived extracellular vesicles increase Foxp3 level and suppressive activity of thymic regulatory T cells. All authors Swatler J, Dudka W, Bugajski L, Brewinska-Olchowik M, Kozlowska E, Piwocka K. Journal Eur J Immunol Abstract Mechanisms driving immunosuppression in chronic myeloid leukemia are mostly unknown. We show that le (show more...)Mechanisms driving immunosuppression in chronic myeloid leukemia are mostly unknown. We show that leukemic extracellular vesicles (EVs) target lymphocytes and amplify suppressive function of thymic regulatory T cells, by driving expression of Foxp3 transcription factor. This could facilitate expansion of leukemic cells outside the bone marrow, leading to blast crisis. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Flotillin1/ Alix/ beta-actin/ TSG101/ HSP70/ CD81 non-EV: Grp78/ TOM20 Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 32D EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified 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) 100 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 140000 Wash: volume per pellet (ml) 55 Wash: time (min) 100 Wash: Rotor Type Type 45 Ti Wash: speed (g) 140000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins Flotillin1/ Alix/ beta-actin/ TSG101/ HSP70/ CD81 Not detected contaminants Grp78/ TOM20 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190104	2/2	Mus musculus	32D	(d)(U)C	Swatler J	2019	78%
Study summary Full title Chronic myeloid leukemia-derived extracellular vesicles increase Foxp3 level and suppressive activity of thymic regulatory T cells. All authors Swatler J, Dudka W, Bugajski L, Brewinska-Olchowik M, Kozlowska E, Piwocka K. Journal Eur J Immunol Abstract Mechanisms driving immunosuppression in chronic myeloid leukemia are mostly unknown. We show that le (show more...)Mechanisms driving immunosuppression in chronic myeloid leukemia are mostly unknown. We show that leukemic extracellular vesicles (EVs) target lymphocytes and amplify suppressive function of thymic regulatory T cells, by driving expression of Foxp3 transcription factor. This could facilitate expansion of leukemic cells outside the bone marrow, leading to blast crisis. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin chronic myeloid leukemia (overexpresssion of BCR-ABL) Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Flotillin1/ Alix/ beta-actin/ TSG101/ HSP70/ CD81 non-EV: Grp78/ TOM20 Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 32D EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified 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) 100 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 140000 Wash: volume per pellet (ml) 55 Wash: time (min) 100 Wash: Rotor Type Type 45 Ti Wash: speed (g) 140000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins Flotillin1/ Alix/ beta-actin/ TSG101/ HSP70/ CD81 Not detected contaminants Grp78/ TOM20 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 135.9 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190055	1/4	Homo sapiens	ARPE-19	DG (d)(U)C	Ferreira JV	2019	78%
Study summary Full title Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis. All authors Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P. Journal PLoS One Abstract Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD63/ Hsc70/ Flotillin1/ GAPDH/ CANX non-EV: Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ARPE-19 EV-harvesting Medium EV-depleted medium 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) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 210000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Size Used subtypes < 200 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Flotillin1/ CD63/ GAPDH/ Hsc70 Not detected EV-associated proteins CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Other particle analysis name(1) RhoB-PE staining Report type Not Reported EV-concentration No

	EV190055	3/4	Homo sapiens	ARPE-19	DG (d)(U)C	Ferreira JV	2019	78%
Study summary Full title Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis. All authors Ferreira JV, Rosa Soares A, Ramalho JS, Ribeiro-Rodrigues T, Máximo C, Zuzarte M, Girão H, Pereira P. Journal PLoS One Abstract Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the ac (show more...)Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD63/ HSC70/ Flotillin1/ GAPDH/ CANX non-EV: Proteomics no EV density (g/ml) 1.15 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ARPE-19 EV-harvesting Medium EV-depleted medium 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) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 120000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 16 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 210000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 38 Pelleting: duration (min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 120000 EV-subtype Distinction between multiple subtypes Size Used subtypes < 200 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Flotillin1/ CD63/ GAPDH/ HSC70 Not detected EV-associated proteins CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Other particle analysis name(1) RhoB-PE staining Report type Not Reported EV-concentration No

	EV190038	2/3	Homo sapiens	Exudative seroma	DG (d)(U)C	García-Silva S	2019	78%
Study summary Full title Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAF V600E mutation. All authors García-Silva S, Benito-Martín A, Sánchez-Redondo S, Hernández-Barranco A, Ximénez-Embún P, Nogués L, Mazariegos MS, Brinkmann K, Amor López A, Meyer L, Rodríguez C, García-Martín C, Boskovic J, Letón R, Montero C, Robledo M, Santambrogio L, Sue Brady M, Szumera-Ciećkiewicz A, Kalinowska I, Skog J, Noerholm M, Muñoz J, Ortiz-Romero PL, Ruano Y, Rodríguez-Peralto JL, Rutkowski P, Peinado H. Journal J Exp Med Abstract Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The asse (show more...)Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse. (hide) EV-METRIC 78% (50th 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 Exudative seroma Sample origin Melanoma patients stage III Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD63/ CD81/ HSP90/ GAPDH/ TRP2/ CD9 non-EV: Proteomics yes EV density (g/ml) Not specified Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Exudative seroma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 50.4 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type Type 50.4 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 8 Sample volume (mL) 0.1 Orientation Top-down Rotor type Type 70.1 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 20 Pelleting: duration (min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ GAPDH/ CD81/ HSP90/ TRP2 Proteomics database ProteomeXchange Consortium Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 148 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 63.6

	EV190028	1/1	Homo sapiens	THP-1	(d)(U)C Filtration	Ramanathan S	2019	78%
Study summary Full title Inflammation potentiates miR-939 expression and packaging into small extracellular vesicles. All authors Ramanathan S, Shenoda BB, Lin Z, Alexander GM, Huppert A, Sacan A, Ajit SK. Journal J Extracell Vesicles Abstract Extracellular RNA in circulation mediates intercellular communication in normal and pathological pro (show more...)Extracellular RNA in circulation mediates intercellular communication in normal and pathological processes. One mode of circulating miRNA transport in bodily fluids is within 30-150 nm small extracellular vesicles (sEVs) or exosomes. Uptake of sEVs can regulate gene expression in recipient cells enabling circulating miRNAs to exert paracrine and systemic effects. Complex regional pain syndrome (CRPS) is a debilitating pain disorder characterized by chronic inflammation. Our previous investigations identified a significant decrease of hsa-miR-939 in whole blood from CRPS patients compared to control; we also observed that overexpression of miR-939 can negatively regulate several proinflammatory genes in vitro. Though downregulated in whole blood, miR-939 was significantly upregulated in sEVs isolated from patient serum. Here we investigated miR-939 packaging into sEVs in vitro under inflammation induced by monocyte chemoattractant protein-1 (MCP-1), a chemokine that is upregulated in CRPS patients. Stimulation of THP-1 monocytes by MCP-1 led to elevated levels of miR-939 in sEVs, which was abrogated using inhibitors of exosome secretion. miRNAs loaded into exosomes largely contain short miRNA sequence motifs called EXOmotifs. Mutation analysis of miR-939 showed that EXOmotif is one of the possible cellular mechanisms responsible for packaging miR-939 into sEVs. We confirmed gene expression changes in recipient cells following the uptake of sEVs enriched in miR-939 using RNA sequencing. Additionally, our data from primary immune cell-derived sEVs of CRPS patients and controls demonstrate that while the relative expression of miR-939 is higher in sEVs derived from B cells, T cells and NK cells relative to monocyte-derived sEVs in controls, only the B cell-derived sEVs showed a significantly higher level of miR-939 in CRPS patients. Differential miRNA sorting into exosomes and its functional impact on recipient cells may contribute to the underlying pathophysiology of CRPS. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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: Alix/ CD81/ TSG101 non-EV: Albumin/ GM130 Proteomics no Show all info Study aim Function/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 25 Wash: time (min) 70 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Detected EV-associated proteins Alix/ CD81/ TSG101 Not detected contaminants GM130/ Albumin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 103.2+/-4.9 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein CD81;CD9 Image type Close-up, Wide-field Report size (nm)

	EV190025	3/4	Homo sapiens	ascites	(d)(U)C Filtration	Czystowska-Kuzmicz, Malgorzata	2019	78%
Study summary Full title Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma All authors Malgorzata Czystowska-Kuzmicz, Anna Sosnowska, Dominika Nowis, Kavita Ramji, Marta Szajnik, Justyna Chlebowska-Tuz, Ewa Wolinska, Pawel Gaj, Magdalena Grazul, Zofia Pilch, Abdessamad Zerrouqi, Agnieszka Graczyk-Jarzynka, Karolina Soroczynska, Szczepan Cierniak, Robert Koktysz, Esther Elishaev, Slawomir Gruca, Artur Stefanowicz, Roman Blaszczyk, Bartlomiej Borek, Anna Gzik, Theresa Whiteside, and Jakub Golab Journal Nat Commun Abstract Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (show more...)Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type ascites Sample origin ovarian cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ ARG1/ EpCAM/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type ascites 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 Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 60 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD63/ TSG101/ ARG1 Not detected contaminants Calnexin Flow cytometry specific beads Detected EV-associated proteins EpCAM/ CD9/ CD81/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125 EV concentration Yes TRPS Report type Mean Reported size (nm) 116 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190025	4/4	Homo sapiens	ovarian cyst fluid	(d)(U)C Filtration	Czystowska-Kuzmicz, Malgorzata	2019	78%
Study summary Full title Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma All authors Malgorzata Czystowska-Kuzmicz, Anna Sosnowska, Dominika Nowis, Kavita Ramji, Marta Szajnik, Justyna Chlebowska-Tuz, Ewa Wolinska, Pawel Gaj, Magdalena Grazul, Zofia Pilch, Abdessamad Zerrouqi, Agnieszka Graczyk-Jarzynka, Karolina Soroczynska, Szczepan Cierniak, Robert Koktysz, Esther Elishaev, Slawomir Gruca, Artur Stefanowicz, Roman Blaszczyk, Bartlomiej Borek, Anna Gzik, Theresa Whiteside, and Jakub Golab Journal Nat Commun Abstract Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (show more...)Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses. (hide) EV-METRIC 78% (50th 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 ovarian cyst fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ ARG1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type ovarian cyst fluid 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 Type 70 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 60 Wash: Rotor Type Type 70 Ti Wash: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins ARG1/ CD63/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 124 EV concentration Yes TRPS Report type Mean Reported size (nm) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190011	1/5	Mus musculus	MC38	(d)(U)C	Cianciaruso C	2019	78%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ Syntenin1/ CD63/ CD81/ GAPDH/ Alix/ vinculin/ actin-beta/ HER2/ CD9/ CD11b non-EV: Calnexin/ Gp96 Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells MC38 EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD81/ vinculin/ GAPDH/ Alix/ TSG101/ Syntenin1/ actin-beta/ CD9/ CD63 Detected contaminants Calnexin/ Gp96 Flow cytometry specific beads Detected EV-associated proteins HER2/ CD11b/ CD9 Flow cytometry Type of Flow cytometry Attune NxT apparatus Hardware adaptation to ~100nm EV's Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep Detected EV-associated proteins HER2 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 150 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190011	3/5	Mus musculus	Bone marrow-derived macrophages	(d)(U)C	Cianciaruso C	2019	78%
Study summary Full title Molecular Profiling and Functional Analysis of Macrophage-Derived Tumor Extracellular Vesicles. All authors Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M Journal Cell Rep Abstract Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: / TSG101/ CD63/ MRC1/ CD81/ GAPDH/ CD68/ Alix/ CD9 non-EV: Gp96 Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Bone marrow-derived macrophages EV-harvesting Medium Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 35 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD81/ MRC1/ GAPDH/ CD63/ TSG101/ CD9/ CD68/ MRC1/ Alix Not detected EV-associated proteins Not detected contaminants Gp96 Flow cytometry Type of Flow cytometry Attune NxT apparatus Hardware adaptation to ~100nm EV's Acquisition settings were optimized for detection of EV populations carrying green, red or near-infrared fluorescence, or combination of those. The conventional blue side scatter (SSC, 488 nm) was rep Detected EV-associated proteins Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 140 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180021	3/4	Homo sapiens	Serum	(d)(U)C Filtration	Bachurski, Daniel	2019	77%
Study summary Full title Extracellular vesicle measurements with nanoparticle tracking analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView All authors Daniel Bachurski ORCID Icon, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi ORCID Icon, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek & Elke Pogge von Strandmann Journal J Extracell Vesicles Abstract The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods t (show more...)The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5–134.3) compared to ZetaView (% DTEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution. (hide) EV-METRIC 77% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 209.7 (pelleting) / 89.2 (washing) Protein markers EV: TSG101/ HSP70/ CD63/ CD9/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 209.7 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63, HSP70, TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-100 Other particle analysis name(1) ExoView Report type Size range/distribution Report size 50-100 EV-concentration No Extra information EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView

	EV180021	4/4	Homo sapiens	L540	(d)(U)C Filtration	Bachurski, Daniel	2019	77%
Study summary Full title Extracellular vesicle measurements with nanoparticle tracking analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView All authors Daniel Bachurski ORCID Icon, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi ORCID Icon, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek & Elke Pogge von Strandmann Journal J Extracell Vesicles Abstract The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods t (show more...)The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5–134.3) compared to ZetaView (% DTEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution. (hide) EV-METRIC 77% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 209.7 (pelleting) / 89.2 (washing) Protein markers EV: TSG101/ HSP70/ CD63/ CD9/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells L540 EV-harvesting Medium Serum free medium Cell viability (%) NA 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) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 209.7 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63, HSP70, TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-400 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150 Other particle analysis name(1) ExoView Report type Size range/distribution Report size 50-100 EV-concentration No Extra information EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView

	EV190051	2/4	Homo sapiens	Serum	(d)(U)C qEV miRCURY	Stefanie Hermann	2019	75%
Study summary Full title Transcriptomic profiling of cell-free and vesicular microRNAs from matched arterial and venous sera All authors Stefanie Hermann, Dominik Buschmann, Benedikt Kirchner, Melanie Borrmann, Florian Brandes, Stefan Kotschote, Michael Bonin, Anja Lindemann, Marlene Reithmair, Gustav Schelling, Michael W. Pfaffl Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) play central physiological and pathophysiological roles in intercellula (show more...)Extracellular vesicles (EVs) play central physiological and pathophysiological roles in intercellular communication. Biomarker studies addressing disorders such as cardiovascular diseases often focus on circulating microRNAs (miRNAs) and may, depending on the type of disease and clinic routine, utilise patient specimens sampled from arterial or venous blood vessels. Thus, it is essential to test whether circulating miRNA profiles depend on the respective sampling site. We assessed potential differences in arterial and venous cell-free miRNA profiles in a cohort of 20 patients scheduled for cardiac surgery. Prior to surgery, blood was simultaneously sampled from the radial artery and the internal jugular vein. After precipitating crude EVs, we performed small RNA Sequencing, which failed to detect significantly regulated miRNAs using stringent filtering criteria for differential expression analysis. Filtering with less strict criteria, we detected four miRNAs slightly upregulated in arterial samples, one of which could be validated by reverse transcription real-time PCR. The applicability of these findings to purified arterial and venous EVs was subsequently tested in a subset of the initial study population. While an additional clean-up step using size-exclusion chromatography seemed to reduce overall miRNA yield compared to crude EV samples, no miRNAs with differential arteriovenous expression were detected. Unsupervised clustering approaches were unable to correctly classify samples drawn from arteries or veins based on miRNAs in either crude or purified preparations. Particle characterisation of crude preparations as well as characterisation of EV markers in purified EVs resulted in highly similar characteristics for arterial and venous samples. With the exception of specific pathologies (e.g. severe pulmonary disorders), arterial versus venous blood sampling should therefore not represent a likely confounder when studying differentially expressed circulating miRNAs. The use of either arterial or venous serum EV samples should result in highly similar data on miRNA expression profiles for the majority of biomarker studies. (hide) EV-METRIC 75% (98th 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 arterial blood, cardiac surgery Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV miRCURY Protein markers EV: Alix/ CD81/ CD63/ Syntenin non-EV: Calnexin/ Albumin/ ApoA1 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW60 Ti Pelleting: speed (g) 200000 Commercial kit qEV;miRCURY Other Name other separation method qEV Other Name other separation method miRCURY Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ CD63/ Syntenin/ CD81 Detected contaminants ApoA1/ Albumin Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 138.54 ± 10.18 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190051	4/4	Homo sapiens	Serum	(d)(U)C qEV miRCURY	Stefanie Hermann	2019	75%
Study summary Full title Transcriptomic profiling of cell-free and vesicular microRNAs from matched arterial and venous sera All authors Stefanie Hermann, Dominik Buschmann, Benedikt Kirchner, Melanie Borrmann, Florian Brandes, Stefan Kotschote, Michael Bonin, Anja Lindemann, Marlene Reithmair, Gustav Schelling, Michael W. Pfaffl Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) play central physiological and pathophysiological roles in intercellula (show more...)Extracellular vesicles (EVs) play central physiological and pathophysiological roles in intercellular communication. Biomarker studies addressing disorders such as cardiovascular diseases often focus on circulating microRNAs (miRNAs) and may, depending on the type of disease and clinic routine, utilise patient specimens sampled from arterial or venous blood vessels. Thus, it is essential to test whether circulating miRNA profiles depend on the respective sampling site. We assessed potential differences in arterial and venous cell-free miRNA profiles in a cohort of 20 patients scheduled for cardiac surgery. Prior to surgery, blood was simultaneously sampled from the radial artery and the internal jugular vein. After precipitating crude EVs, we performed small RNA Sequencing, which failed to detect significantly regulated miRNAs using stringent filtering criteria for differential expression analysis. Filtering with less strict criteria, we detected four miRNAs slightly upregulated in arterial samples, one of which could be validated by reverse transcription real-time PCR. The applicability of these findings to purified arterial and venous EVs was subsequently tested in a subset of the initial study population. While an additional clean-up step using size-exclusion chromatography seemed to reduce overall miRNA yield compared to crude EV samples, no miRNAs with differential arteriovenous expression were detected. Unsupervised clustering approaches were unable to correctly classify samples drawn from arteries or veins based on miRNAs in either crude or purified preparations. Particle characterisation of crude preparations as well as characterisation of EV markers in purified EVs resulted in highly similar characteristics for arterial and venous samples. With the exception of specific pathologies (e.g. severe pulmonary disorders), arterial versus venous blood sampling should therefore not represent a likely confounder when studying differentially expressed circulating miRNAs. The use of either arterial or venous serum EV samples should result in highly similar data on miRNA expression profiles for the majority of biomarker studies. (hide) EV-METRIC 75% (98th 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 venous blood, cardiac surgery Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C qEV miRCURY Protein markers EV: Alix/ CD81/ CD63/ Syntenin non-EV: Calnexin/ Albumin/ ApoA1 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW60 Ti Pelleting: speed (g) 200000 Commercial kit qEV;miRCURY Other Name other separation method qEV Other Name other separation method miRCURY Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ Syntenin/ CD63/ CD81 Detected contaminants ApoA1/ Albumin Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 147.54 ± 6.84 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190040	4/12	Homo sapiens	HEK293T	DG UF	Geeurickx E	2019	75%
Study summary Full title The generation and use of recombinant extracellular vesicles as biological reference material. All authors Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A. Journal Nat Commun Abstract Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide) EV-METRIC 75% (96th 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 gag-EGFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG UF Protein markers EV: TSG101/ CD81/ Alix/ Flotillin1/ CD9/ syntenin-1 non-EV: Proteomics yes EV density (g/ml) 1.086-1.119 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium Cell viability (%) 96 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Used subtypes 1.076 1.088 g/ml Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ Alix/ CD9/ TSG101/ syntenin-1/ CD81 Proteomics database Yes: Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190039	2/3	Homo sapiens	Blood plasma	DG (d)(U)C SEC SEC (non-commercial) UF	Everaert, Celine	2019	75%
Study summary Full title Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles All authors Celine Everaert, Hetty Helsmoortel, Anneleen Decock, Eva Hulstaert, Ruben Van Paemel, Kimberly Verniers, Justine Nuytens 1 2 , Jasper Anckaert 1 2 , Nele Nijs, Joeri Tulkens, Bert Dhondt, An Hendrix, Pieter Mestdagh, Jo Vandesompele Journal Scientific Reports Abstract RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human bioflui (show more...)RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C SEC SEC (non-commercial) UF Protein markers EV: Flotillin1/ CD9 non-EV: ApoA-1 Proteomics no EV density (g/ml) 1.1 Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1091 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Other Name other separation method SEC (non-commercial) Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ CD9 Detected contaminants ApoA-1 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-200 EM EM-type Transmission-EM Image type Close-up

	EV190018	1/8	Homo sapiens	MKN45	(d)(U)C Filtration	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: Alix/ CD63/ CD81/ Proteins regarded in the experiment as potentially EV-associated and not detected in the EVs/ HSP70/ CD9/ Syntenin-1 non-EV: Cytochrome C/ Albumin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 960 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 120 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins CD9/ CD63/ Syntenin-1/ HSP70/ Alix/ CD81 Not detected EV-associated proteins Proteins regarded in the experiment as potentially EV-associated and not detected in the EVs Detected contaminants Albumin Not detected contaminants Cytochrome C Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140.97 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	2/8	Homo sapiens	MKN45	(d)(U)C Filtration Total Exosome Isolation	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Total Exosome Isolation Protein markers EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1 non-EV: Cytochrome C/ Albumin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 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 Filtration steps 0.22µm or 0.2µm Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins CD63/ Syntenin-1/ HSP70/ CD81 Not detected EV-associated proteins CD9/ Alix Detected contaminants Albumin Not detected contaminants Cytochrome C Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148.3 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	4/8	Homo sapiens	MKN45	(d)(U)C Filtration qEV	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration qEV Protein markers EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1 non-EV: Albumin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Syntenin-1/ CD9/ CD63/ HSP70/ CD81 Not detected EV-associated proteins Alix Detected contaminants Albumin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 138.37 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	5/8	Homo sapiens	MKN45	(d)(U)C Filtration	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 COSMC KO Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1 non-EV: Albumin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 960 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 120 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Syntenin-1/ CD9/ CD63/ HSP70/ Alix/ CD81 Detected contaminants Albumin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 141.43 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	6/8	Homo sapiens	MKN45	(d)(U)C Filtration Total Exosome Isolation	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 COSMC KO Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Total Exosome Isolation Protein markers EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1 non-EV: Albumin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium 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 Filtration steps 0.22µm or 0.2µm Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Alix/ Syntenin-1/ CD63/ HSP70/ CD81 Not detected EV-associated proteins CD9 Detected contaminants Albumin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 152.5 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	8/8	Homo sapiens	MKN45	(d)(U)C Filtration qEV	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 COSMC KO Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration qEV Protein markers EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1 non-EV: Albumin Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Alix/ Syntenin-1/ CD9/ CD63/ HSP70/ CD81 Detected contaminants Albumin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148.33 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190007	2/5	Homo sapiens	Urine	(d)(U)C Norgen Biotek Urine Exosome Purification Kit UF	Mussack V	2019	75%
Study summary Full title Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing-Enabling robust and non-invasive biomarker research. All authors Mussack V, Wittmann G, Pfaffl MW. Journal Biomol Detect Quanti Abstract Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication th (show more...)Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study. (hide) EV-METRIC 75% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Norgen Biotek Urine Exosome Purification Kit UF Protein markers EV: TSG101/ CD63/ CD81/ Alix/ Syntenin/ EPCAM/ HSP70/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit Other;Norgen Biotek Urine Exosome Purification Kit Other Name other separation method Norgen Biotek Urine Exosome Purification Kit Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix Not detected EV-associated proteins HSP70/ CD81/ Syntenin/ EPCAM/ TSG101/ CD63/ CD9 Not detected contaminants Calnexin/ Tamm-Horsfall protein Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 130 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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