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You searched for: EV190108 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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
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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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
Sample Condition
Metastatic melanoma
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
20
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
16500
Density gradient
Density medium
Iodixanol
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: 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
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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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
Sample Condition
Metastatic melanoma
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
20
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
16500
Density gradient
Density medium
Iodixanol
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: 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
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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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
Sample Condition
Metastatic melanoma
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
150
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
11800
Density gradient
Density medium
Iodixanol
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: 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
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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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
Sample Condition
Metastatic melanoma
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
150
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
118000
Density gradient
Density medium
Iodixanol
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: 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
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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(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
Sample Condition
Metastatic melanoma
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
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: 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
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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(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
Sample Condition
Metastatic melanoma
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
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: 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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