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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

	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

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EV-TRACK ID	EV190108
species	Homo sapiens
sample type	Tumour tissue
condition	Metastatic melanoma
separation protocol	DG (d)(U)C	DG (d)(U)C	DG (d)(U)C	DG (d)(U)C	(d)(U)C	(d)(U)C
vesicle related term	Other Large low density EVs (Large LD EVs)	Other Large high density EVs (Large HD EVs)	Other Small low density EVs (Small LD EVs)	Other Small high density EVs (Small HD EVs)	Other Large EVs (Large EVs)	Other Small EVs (Small EVs)
Exp. nr.	3	4	5	6	1	2
EV-METRIC %	100	100	100	100	78	78