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You searched for: EV200073 (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
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV200073 2/4 Homo sapiens Solid Tissue (d)(U)C
Size-exclusion chromatopraphy (IZON)
DC
DG
Bordas, Marie 2020 88%

Study summary

Full title
All authors
Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert
Journal
Int J Mol Sci
Abstract
Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide)
EV-METRIC
88% (75th 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
Solid Tissue
Sample origin
CLL
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
Size-exclusion chromatopraphy (IZON)
DC
DG
Protein markers
EV: TSG101/ CD81/ Flotillin1/ CD9
non-EV: cytochrome C/ GM130/ Calreticulin
Proteomics
no
EV density (g/ml)
1.12
Show all info
Study aim
Function/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Solid Tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
2
Lowest density fraction
0%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10.5
Sample volume (mL)
7
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
100
Duration (min)
120
Fraction volume (mL)
2.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density cushion
Density medium
Sucrose
Other
Name other separation method
Size-exclusion chromatopraphy (IZON)
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101/ CD81
Detected contaminants
Calreticulin
Not detected contaminants
cytochrome C/ GM130
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.00E+10
EM
EM-type
Immuno-EM/ Transmission-EM
EM protein
Other;HLA-DR
Image type
Close-up
EV200073 4/4 Mus musculus Solid Tissue (d)(U)C
Size-exclusion chromatopraphy (IZON)
DC
DG
Bordas, Marie 2020 88%

Study summary

Full title
All authors
Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert
Journal
Int J Mol Sci
Abstract
Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide)
EV-METRIC
88% (75th 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
Solid Tissue
Sample origin
CLL
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
Size-exclusion chromatopraphy (IZON)
DC
DG
Protein markers
EV: Alix/ TSG101/ Flotillin1
non-EV: ATPA5/ Calreticulin
Proteomics
no
EV density (g/ml)
1.12
Show all info
Study aim
Function/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Solid Tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
2
Lowest density fraction
0%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10.5
Sample volume (mL)
7
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
100
Duration (min)
120
Fraction volume (mL)
2.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density cushion
Density medium
Sucrose
Other
Name other separation method
Size-exclusion chromatopraphy (IZON)
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ TSG101
Detected contaminants
Calreticulin
Not detected contaminants
ATPA5
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
140
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 7.00E+09
EM
EM-type
Transmission-EM
Image type
Close-up
EV200073 1/4 Homo sapiens Solid Tissue (d)(U)C
Size-exclusion chromatopraphy (IZON)
DC
SEC (IZON)
Bordas, Marie 2020 63%

Study summary

Full title
All authors
Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert
Journal
Int J Mol Sci
Abstract
Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide)
EV-METRIC
63% (25th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Solid Tissue
Sample origin
CLL
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
Size-exclusion chromatopraphy (IZON)
DC
SEC (IZON)
Protein markers
EV: TSG101/ CD81/ Flotillin1/ CD9
non-EV: Cytochrome C/ GM130/ Calreticulin
Proteomics
no
Show all info
Study aim
Function/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Solid Tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
No
Density cushion
Density medium
Sucrose
Other
Name other separation method
Size-exclusion chromatopraphy (IZON)
Other
Name other separation method
SEC (IZON)
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101/ CD81
Detected contaminants
Calreticulin
Not detected contaminants
Cytochrome C/ GM130
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.00E+10
EM
EM-type
Immuno-EM/ Transmission-EM
EM protein
Other;HLA-DR
Image type
Close-up
EV200073 3/4 Mus musculus Solid Tissue (d)(U)C
Size-exclusion chromatopraphy (IZON)
DC
SEC (IZON)
Bordas, Marie 2020 63%

Study summary

Full title
All authors
Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert
Journal
Int J Mol Sci
Abstract
Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide)
EV-METRIC
63% (25th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Solid Tissue
Sample origin
CLL
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
Size-exclusion chromatopraphy (IZON)
DC
SEC (IZON)
Protein markers
EV: Alix/ TSG101/ CD81
non-EV: ATPA5/ Calreticulin
Proteomics
no
Show all info
Study aim
Function/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Solid Tissue
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
No
Density cushion
Density medium
Sucrose
Other
Name other separation method
Size-exclusion chromatopraphy (IZON)
Other
Name other separation method
SEC (IZON)
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ CD81
Detected contaminants
Calreticulin
Not detected contaminants
ATPA5
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
145
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 3.50E+10
EM
EM-type
Transmission-EM
Image type
Close-up
1 - 4 of 4
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200073
species
Homo sapiens
Mus musculus
Homo sapiens
Mus musculus
sample type
Solid Tissue
Solid Tissue
Solid Tissue
Solid Tissue
condition
CLL
CLL
CLL
CLL
separation protocol
(d)(U)C
Size-exclusion chromatopraphy (IZON)
DC
DG
(d)(U)C
Size-exclusion chromatopraphy (IZON)
DC
DG
(d)(U)C
Size-exclusion chromatopraphy (IZON)
DC
SEC (IZON)
(d)(U)C
Size-exclusion chromatopraphy (IZON)
DC
SEC (IZON)
Exp. nr.
2
4
1
3
EV-METRIC %
88
88
63
63