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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 code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210118 1/4 Bos taurus cheese manufacturing byproducts DG
tangential flow filtration
Filtration
Sukreet, Sonal 2021 100%

Study summary

Full title
All authors
Sonal Sukreet, Camila Pereira Braga, Thuy T. An, Jiri Adamec, Juan Cui, Benjamin Trible, Janos Zempleni
Journal
Journal of Dairy Science
Abstract
Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as (show more...)Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV. (hide)
EV-METRIC
100% (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
cheese manufacturing byproducts
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
tangential flow filtration
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9
non-EV: Integrin-beta/ Histone H3/ ApoB
Proteomics
yes
EV density (g/ml)
1.255
Show all info
Study aim
New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
cheese manufacturing byproducts
Separation Method
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
M.W. >100 kDa
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101/ HSP70
Not detected EV-associated proteins
CD81/ CD63/ CD9/ Alix
Detected contaminants
Histone H3/ Integrin-beta/ ApoB
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
1108+/-138
NTA
Report type
Mean
Reported size (nm)
102.3+/-6.7
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.12+/-0.525E09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210118 2/4 Bos taurus cheese manufacturing byproducts DG
tangential flow filtration
Filtration
Sukreet, Sonal 2021 100%

Study summary

Full title
All authors
Sonal Sukreet, Camila Pereira Braga, Thuy T. An, Jiri Adamec, Juan Cui, Benjamin Trible, Janos Zempleni
Journal
Journal of Dairy Science
Abstract
Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as (show more...)Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV. (hide)
EV-METRIC
100% (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
cheese manufacturing byproducts
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
tangential flow filtration
Filtration
Protein markers
EV: CD81/ Alix/ CD63/ CD9/ HSP70
non-EV: Integrin-beta/ Histone H3/ ApoB
Proteomics
yes
EV density (g/ml)
1.255
Show all info
Study aim
New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
cheese manufacturing byproducts
Separation Method
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
M.W. 50 kDa to 100 kDa
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
HSP70
Not detected EV-associated proteins
CD81/ CD63/ CD9/ Alix
Not detected contaminants
Histone H3/ Integrin-beta/ ApoB
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
45+/-17
NTA
Report type
Mean
Reported size (nm)
80.9+/-4.3
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.06+/-0.22E09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210118 3/4 Bos taurus cheese manufacturing byproducts DG
tangential flow filtration
Filtration
Sukreet, Sonal 2021 100%

Study summary

Full title
All authors
Sonal Sukreet, Camila Pereira Braga, Thuy T. An, Jiri Adamec, Juan Cui, Benjamin Trible, Janos Zempleni
Journal
Journal of Dairy Science
Abstract
Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as (show more...)Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV. (hide)
EV-METRIC
100% (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
cheese manufacturing byproducts
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
tangential flow filtration
Filtration
Protein markers
EV: CD81/ Alix/ CD63/ CD9/ HSP70
non-EV: Integrin-beta/ Histone H3/ ApoB
Proteomics
yes
EV density (g/ml)
1.255
Show all info
Study aim
New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
cheese manufacturing byproducts
Separation Method
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
M.W. >50 kDa
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
HSP70
Not detected EV-associated proteins
CD81/ CD63/ CD9/ Alix
Not detected contaminants
Histone H3/ Integrin-beta/ ApoB
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
157+/-122
NTA
Report type
Mean
Reported size (nm)
90.8
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.44+/-0.144E09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210118 4/4 Bos taurus skim milk DG
(d)(U)C
Sukreet, Sonal 2021 100%

Study summary

Full title
All authors
Sonal Sukreet, Camila Pereira Braga, Thuy T. An, Jiri Adamec, Juan Cui, Benjamin Trible, Janos Zempleni
Journal
Journal of Dairy Science
Abstract
Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as (show more...)Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV. (hide)
EV-METRIC
100% (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
skim milk
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/ HSP70/ CD9
non-EV: Integrin-beta/ Histone H3/ ApoB
Proteomics
yes
EV density (g/ml)
1.255
Show all info
Study aim
New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
skim milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Between 50,000 g and 100,000 g
Pelleting: time(min)
90
Pelleting: rotor type
F37L-8x100
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
F37L-8x100
Wash: speed (g)
120000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101/ HSP70/ CD81
Not detected contaminants
Histone H3/ Integrin-beta/ ApoB
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
115+/-31
NTA
Report type
Mean
Reported size (nm)
106.6
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.42+/-0.0536E14
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200099 1/8 Bos taurus milk (d)(U)C
DG
Kleinjan, Marije 2021 100%

Study summary

Full title
All authors
Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben
Journal
J Nutr
Abstract
Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide)
EV-METRIC
100% (88th 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
milk
Sample origin
raw
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
DG
Protein markers
EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9
non-EV: beta-lactoglobulin/ beta-casein
Proteomics
no
EV density (g/ml)
1.20-1.24
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.20-1.24
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx cell sorter (BD Biosciences, San Jose, CA, USA)
Hardware adjustment
High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles
Calibration bead size
0.1
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
200
EV200099 2/8 Bos taurus milk (d)(U)C
DG
Kleinjan, Marije 2021 100%

Study summary

Full title
All authors
Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben
Journal
J Nutr
Abstract
Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide)
EV-METRIC
100% (88th 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
milk
Sample origin
raw
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
DG
Protein markers
EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9
non-EV: beta-lactoglobulin/ beta-casein
Proteomics
no
EV density (g/ml)
1.18-1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.18-1.13
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx cell sorter (BD Biosciences, San Jose, CA, USA)
Hardware adjustment
High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
200
EV200099 3/8 Bos taurus milk (d)(U)C
DG
Kleinjan, Marije 2021 100%

Study summary

Full title
All authors
Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben
Journal
J Nutr
Abstract
Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide)
EV-METRIC
100% (88th 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
milk
Sample origin
pasteurized
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
DG
Protein markers
EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9
non-EV: beta-lactoglobulin/ beta-casein
Proteomics
no
EV density (g/ml)
1.20-1.24
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.20-1.24
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx cell sorter (BD Biosciences, San Jose, CA, USA)
Hardware adjustment
High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles
Calibration bead size
0.1
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
200
EV200099 4/8 Bos taurus milk (d)(U)C
DG
Kleinjan, Marije 2021 100%

Study summary

Full title
All authors
Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben
Journal
J Nutr
Abstract
Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide)
EV-METRIC
100% (88th 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
milk
Sample origin
pasteurized
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
DG
Protein markers
EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9
non-EV: beta-lactoglobulin/ beta-casein
Proteomics
no
EV density (g/ml)
1.18-1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.18-1.13
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx cell sorter (BD Biosciences, San Jose, CA, USA)
Hardware adjustment
High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
200
EV200099 5/8 Bos taurus milk (d)(U)C
DG
Kleinjan, Marije 2021 100%

Study summary

Full title
All authors
Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben
Journal
J Nutr
Abstract
Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide)
EV-METRIC
100% (88th 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
milk
Sample origin
pasteurized and homogenized
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
DG
Protein markers
EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9
non-EV: beta-lactoglobulin/ beta-casein
Proteomics
no
EV density (g/ml)
1.20-1.24
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.20-1.24
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx cell sorter (BD Biosciences, San Jose, CA, USA)
Hardware adjustment
High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles
Calibration bead size
0.1
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
200
EV200099 6/8 Bos taurus milk (d)(U)C
DG
Kleinjan, Marije 2021 100%

Study summary

Full title
All authors
Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben
Journal
J Nutr
Abstract
Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide)
EV-METRIC
100% (88th 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
milk
Sample origin
pasteurized and homogenized
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
DG
Protein markers
EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9
non-EV: beta-lactoglobulin/ beta-casein
Proteomics
no
EV density (g/ml)
1.18-1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.18-1.13
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx cell sorter (BD Biosciences, San Jose, CA, USA)
Hardware adjustment
High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
200
EV200099 7/8 Bos taurus milk (d)(U)C
DG
Kleinjan, Marije 2021 100%

Study summary

Full title
All authors
Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben
Journal
J Nutr
Abstract
Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide)
EV-METRIC
100% (88th 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
milk
Sample origin
UHT
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
DG
Protein markers
EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9
non-EV: beta-lactoglobulin/ beta-casein
Proteomics
no
EV density (g/ml)
1.20-1.24
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.20-1.24
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx cell sorter (BD Biosciences, San Jose, CA, USA)
Hardware adjustment
High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles
Calibration bead size
0.1
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
200
EV200099 8/8 Bos taurus milk (d)(U)C
DG
Kleinjan, Marije 2021 100%

Study summary

Full title
All authors
Marije Kleinjan, Martijn Jc van Herwijnen, Sten Fwm Libregts, Rj Joost van Neerven, Anouk L Feitsma, Marca Hm Wauben
Journal
J Nutr
Abstract
Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellul (show more...)Background: Bovine milk contains extracellular vesicles (EVs), which act as mediators of intercellular communication by regulating the recipients’ cellular processes via their selectively incorporated bioactive molecules. Because some of these EV components are evolutionarily conserved, EVs present in commercial milk might have the potential to regulate cellular processes in human consumers. Objectives: Because commercial milk is subjected to industrial processing, we investigated its effect on the number and integrity of isolated milk EVs and their bioactive components. For this, we compared EVs isolated from raw bovine milk with EVs isolated from different types of commercial milk, including pasteurized milk, either homogenized or not, and ultra heat treated (UHT) milk. Methods: EVs were separated from other milk components by differential centrifugation, followed by density gradient ultracentrifugation. EVs from different milk types were compared by single-particle high-resolution fluorescence-based flow cytometry to determine EV numbers, Cryo-electron microscopy to visualize EV integrity and morphology, western blot analysis to investigate EV-associated protein cargo, and RNA analysis to assess total small RNA concentration and milk-EV-specific microRNA expression. Results: In UHT milk, we could not detect intact EVs. Interestingly, although pasteurization (irrespective of homogenization) did not affect mean ± SD EV numbers (3.4 × 108 ± 1.2 × 108–2.8 × 108 ± 0.3 × 107 compared with 3.1 × 108 ± 1.2 × 108 in raw milk), it affected EV integrity and appearance, altered their protein signature, and resulted in a loss of milk-EV-associated RNAs (from 40.2 ± 3.4 ng/μL in raw milk to 17.7 ± 5.4–23.3 ± 10.0 mg/μL in processed milk, P < 0.05). Conclusions: Commercial milk, that has been heated by either pasteurization or UHT, contains fewer or no intact EVs, respectively. Although most EVs seemed resistant to pasteurization based on particle numbers, their integrity was affected and their molecular composition was altered. Thus, the possible transfer of bioactive components via bovine milk EVs to human consumers is likely diminished or altered in heat-treated commercial milk. (hide)
EV-METRIC
100% (88th 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
milk
Sample origin
UHT
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
DG
Protein markers
EV: MFG-E8/ CD63/ TSG101/ Flotillin1/ CD9
non-EV: beta-lactoglobulin/ beta-casein
Proteomics
no
EV density (g/ml)
1.18-1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
1.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.18-1.13
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx cell sorter (BD Biosciences, San Jose, CA, USA)
Hardware adjustment
High-resolution flow cytometric analysis of PKH67-stained samples was performed on a BD Influx cell sorter (BD Biosciences, San Jose, CA, USA) that was dedicated and optimized for detection of submicron-sized particles
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
200
EV200010 4/4 Homo sapiens Blood plasma DG
(d)(U)C
SEC
Kuypers, Sören 2021 100%

Study summary

Full title
All authors
Sören Kuypers, Nick Smisdom, Isabel Pintelon, Jean-Pierre Timmermans, Marcel Ameloot, Luc Michiels, Jelle Hendrix, Baharak Hosseinkhani
Journal
Small
Abstract
Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell (show more...)Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and 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
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
Protein markers
EV: ICAM/ CD63/ CD9/ ANXA2
non-EV: APOA1
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
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)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 28.1
Speed (g)
100000
Duration (min)
1451
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1-6
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ ANXA2
Not detected contaminants
APOA1
Detected EV-associated proteins
CD9/ CD63/ ICAM
Not detected contaminants
APOA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
100-200
EV190096 1/2 Bos taurus skim milk acetic acid treatment
DG
(d)(U)C
Filtration
Mukhopadhya, Anindya 2021 100%

Study summary

Full title
All authors
Anindya Mukhopadhya, Jessie Santoro, Barry Moran, Zivile Useckaite, Lorraine O'Driscoll
Journal
Food Chem.
Abstract
Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves (show more...)Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM. (hide)
EV-METRIC
100% (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
skim milk
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
acetic acid treatment
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ HLADR/ ADAM10/ CD9
non-EV: Actinin4
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
skim milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
2.33
Orientation
Bottom-up
Rotor type
Type 70.1Ti
Speed (g)
186000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
9
Pelleting: duration (min)
90
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.45µm > x > 0.22µm,
Other
Name other separation method
acetic acid treatment
Other
Name other separation method
isoelectric precipitation
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ TSG101
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Actinin4
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II Flow Cytometer
Calibration bead size
none
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLADR
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
160
EV concentration
Yes
EV190096 2/2 Bos taurus powdered infant milk formula acetic acid treatment
DG
(d)(U)C
Filtration
Mukhopadhya, Anindya 2021 100%

Study summary

Full title
All authors
Anindya Mukhopadhya, Jessie Santoro, Barry Moran, Zivile Useckaite, Lorraine O'Driscoll
Journal
Food Chem.
Abstract
Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves (show more...)Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM. (hide)
EV-METRIC
100% (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
powdered infant milk formula
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
acetic acid treatment
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ HLADR/ ADAM10/ CD9
non-EV: Actinin4
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
powdered infant milk formula
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
2.33
Orientation
Bottom-up
Rotor type
Type 70.1Ti
Speed (g)
186000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
9
Pelleting: duration (min)
90
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.45µm > x > 0.22µm,
Other
Name other separation method
acetic acid treatment
Other
Name other separation method
isoelectric precipitation
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ TSG101
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Actinin4
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II Flow Cytometer
Calibration bead size
none
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLADR
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
175
EV concentration
Yes
EV200159 2/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 89%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide)
EV-METRIC
89% (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
CD63-mCherry
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Alix/ Flotillin1/ CD9/ mcherry
non-EV: Lamin B1
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
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: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ mCherry/ Alix/ CD81
Not detected contaminants
Lamin B1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
particles per milliliter of final volume of sample;Yes, other: 5,00E+13
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
56
EV200159 3/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 89%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide)
EV-METRIC
89% (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
CD63-FLuc
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Alix/ Flotillin1/ CD9
non-EV: Lamin B1
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
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: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ Alix/ Firely luciferase/ CD9/ CD63/ CD81
Not detected contaminants
Lamin B1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
particles per milliliter of final volume of sample;Yes, other: 1,50E+13
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
80
EV200157 5/10 Homo sapiens Cell culture supernatant (d)(U)C
SEC (non-commercial)
Polymer-based precipitation
DG
Martínez-Greene, Juan A 2021 89%

Study summary

Full title
All authors
Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez
Journal
J Extracell Vesicles
Abstract
The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide)
EV-METRIC
89% (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
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Polymer-based precipitation
Density gradient
Protein markers
EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5
non-EV: Albumin
Proteomics
yes
EV density (g/ml)
1.08-1.15
Show all info
Study aim
New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-468
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
1.50E+06
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
39
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
118000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
30%
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
2.5
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
200000
Duration (min)
60
Fraction volume (mL)
0.49
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.8
Pelleting: duration (min)
39
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
118000
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
EV-subtype
Distinction between multiple subtypes
SEC fraction
Used subtypes
F5-10
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5
Detected contaminants
Albumin
Proteomics database
Yes: ProteomeXchange
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
148.9
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.13E+11
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200157 6/10 Homo sapiens Cell culture supernatant (d)(U)C
SEC (non-commercial)
Polymer-based precipitation
DG
Martínez-Greene, Juan A 2021 89%

Study summary

Full title
All authors
Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez
Journal
J Extracell Vesicles
Abstract
The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide)
EV-METRIC
89% (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
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Polymer-based precipitation
Density gradient
Protein markers
EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5
non-EV: Albumin
Proteomics
yes
EV density (g/ml)
1.08-1.15
Show all info
Study aim
New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-468
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
1.50E+06
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
39
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
118000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
30%
Total gradient volume, incl. sample (mL)
5
Sample volume (mL)
2.5
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
200000
Duration (min)
60
Fraction volume (mL)
0.49
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.8
Pelleting: duration (min)
39
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
118000
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
EV-subtype
Distinction between multiple subtypes
SEC fraction
Used subtypes
F11-16
Characterization: Protein analysis
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ ANXA2/ ANXA5
Not detected EV-associated proteins
Alix/ TSG101
Detected contaminants
Albumin
Proteomics database
Yes: ProteomeXchange
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
124
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.29E+11
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200081 1/4 Homo sapiens Blood plasma DG
SEC (non-commercial)
Vergauwen, Glenn 2021 88%

Study summary

Full title
All authors
Glenn Vergauwen, Joeri Tulkens, Cláudio Pinheiro, Francisco Avila Cobos, Sándor Dedeyne, Marie-Angélique De Scheerder, Linos Vandekerckhove, Francis Impens, Ilkka Miinalainen, Geert Braems, Kris Gevaert, Pieter Mestdagh, Jo Vandesompele, Hannelore Denys, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biolog (show more...)Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV. (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
Density gradient
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Flotillin1/ CD9
non-EV: APOB/ APOA1
Proteomics
no
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
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
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1
Not detected contaminants
APOA1
ELISA
Antibody details provided?
No
Detected EV-associated proteins
CD9
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-250
EV210180 1/2 Homo sapiens Cell culture supernatant (d)(U)C Santos, Mark F 2021 78%

Study summary

Full title
All authors
Mark F. Santos, Germana Rappa, Jana Karbanová, Simona Fontana, Maria Antonietta Di Bella, Marshall R. Pope, Barbara Parrino, Stella Maria Cascioferro, Giulio Vistoli, Patrizia Diana, Girolamo Cirrincione, Goffredo O. Arena, Gyunghwi Woo, Kevin Huang, Tony Huynh, Marta Moschetti, Riccardo Alessandro, Denis Corbeil, Aurelio Lorico
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pat (show more...)Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3–VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells. (hide)
EV-METRIC
78% (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
Protein markers
EV: CD63/ CD81/ Alix/ CD9
non-EV: Histone H1/ 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
SW620
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
Equal to or above 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
200000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Alix/ CD81
Not detected contaminants
Histone H1/ Calnexin
Detected EV-associated proteins
CD9/ CD63/ CD81
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
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
60-130
EV210180 2/2 Homo sapiens Cell culture supernatant (d)(U)C Santos, Mark F 2021 78%

Study summary

Full title
All authors
Mark F. Santos, Germana Rappa, Jana Karbanová, Simona Fontana, Maria Antonietta Di Bella, Marshall R. Pope, Barbara Parrino, Stella Maria Cascioferro, Giulio Vistoli, Patrizia Diana, Girolamo Cirrincione, Goffredo O. Arena, Gyunghwi Woo, Kevin Huang, Tony Huynh, Marta Moschetti, Riccardo Alessandro, Denis Corbeil, Aurelio Lorico
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pat (show more...)Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3–VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells. (hide)
EV-METRIC
78% (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
CD9-GFP expression
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: CD63/ CD81/ Alix/ CD9
non-EV: Histone H1/ 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
FEMX-I
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
Equal to or above 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
200000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Alix/ CD81
Not detected contaminants
Histone H1/ Calnexin
Detected EV-associated proteins
CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
120
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.00E+10
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
40-100
EV210143 1/6 Homo sapiens Cell culture supernatant (d)(U)C Martinez-Pacheco, Sarai 2021 78%

Study summary

Full title
All authors
Sarai Martinez-Pacheco and Lorraine O’Driscoll
Journal
Cancers
Abstract
To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide)
EV-METRIC
78% (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
Protein markers
EV: CD63/ CD9/ Syntenin
non-EV: Calnexin/ GRP94
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
SKBR3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
94
Cell count
1.90E+08
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: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
39
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield
Yes, per million cells 0.09
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Syntenin
Not detected contaminants
Calnexin/ GRP94
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
125.7
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2296296296
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210143 2/6 Homo sapiens Cell culture supernatant PEG precipitation
(d)(U)C
Filtration
Martinez-Pacheco, Sarai 2021 78%

Study summary

Full title
All authors
Sarai Martinez-Pacheco and Lorraine O’Driscoll
Journal
Cancers
Abstract
To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide)
EV-METRIC
78% (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
PEG precipitation
(d)(U)C
Filtration
Protein markers
EV: CD63/ CD9/ Syntenin
non-EV: Calnexin/ GRP94
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
SKBR3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
94
Cell count
1.90E+08
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: time(min)
130
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield
Yes, per cell 0.14
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Syntenin
Not detected contaminants
Calnexin/ GRP94
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
110.2
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 6314814815
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210143 3/6 Homo sapiens Cell culture supernatant (d)(U)C Martinez-Pacheco, Sarai 2021 78%

Study summary

Full title
All authors
Sarai Martinez-Pacheco and Lorraine O’Driscoll
Journal
Cancers
Abstract
To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide)
EV-METRIC
78% (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
Protein markers
EV: CD63/ CD9/ Syntenin
non-EV: Calnexin/ GRP94
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
HCC1954
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
93
Cell count
2.10E+08
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: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
39
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield
Yes, per cell 0.03
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ Syntenin
Not detected contaminants
Calnexin/ GRP94
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
129.4
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 7703703703
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210143 4/6 Homo sapiens Cell culture supernatant PEG precipitation
(d)(U)C
Filtration
Martinez-Pacheco, Sarai 2021 78%

Study summary

Full title
All authors
Sarai Martinez-Pacheco and Lorraine O’Driscoll
Journal
Cancers
Abstract
To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide)
EV-METRIC
78% (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
PEG precipitation
(d)(U)C
Filtration
Protein markers
EV: CD63/ CD9/ Syntenin
non-EV: Calnexin/ GRP94
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
HCC1954
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
93
Cell count
2.10E+08
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: time(min)
130
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield
Yes, per cell 0.1
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin/ CD9/ CD63
Not detected contaminants
Calnexin/ GRP94
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
119.3
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 8518481482
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210143 5/6 Homo sapiens Cell culture supernatant (d)(U)C Martinez-Pacheco, Sarai 2021 78%

Study summary

Full title
All authors
Sarai Martinez-Pacheco and Lorraine O’Driscoll
Journal
Cancers
Abstract
To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide)
EV-METRIC
78% (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
Protein markers
EV: CD63/ CD9/ Syntenin
non-EV: Calnexin/ GRP94
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
EFM192A
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
98
Cell count
3.60E+08
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: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
39
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield
Yes, per cell 0.01
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin/ CD63/ CD81
Not detected contaminants
Calnexin/ GRP94
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
131.7
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 727777778
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210143 6/6 Homo sapiens Cell culture supernatant PEG precipitation
(d)(U)C
Filtration
Martinez-Pacheco, Sarai 2021 78%

Study summary

Full title
All authors
Sarai Martinez-Pacheco and Lorraine O’Driscoll
Journal
Cancers
Abstract
To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, (show more...)To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes (hide)
EV-METRIC
78% (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
PEG precipitation
(d)(U)C
Filtration
Protein markers
EV: CD63/ CD9/ Syntenin
non-EV: Calnexin/ GRP94
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
EFM192A
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
98
Cell count
3.60E+08
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: time(min)
130
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield
Yes, per cell 0.11
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin/ CD63/ CD81
Not detected contaminants
Calnexin/ GRP94
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
117.7
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 5018518518
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210126 1/9 Homo sapiens Cell culture supernatant (d)(U)C Silva, Andreia;Lázaro-Ibáñez, Elisa 2021 78%

Study summary

Full title
All authors
Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker
Journal
J Extracell Vesicles
Abstract
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide)
EV-METRIC
78% (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
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ Flotillin1/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
80
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
100
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD63/ GFP/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
CD9
Detected contaminants
calnexin
Flow cytometry
Type of Flow cytometry
NanoAnalyzer N30 (nanoFCM)
Calibration bead size
0.068; 0.091; 0.113; 0.155
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoAnalyzer N30 (nanoFCM)
Hardware adjustment
Calibration bead size
0.068; 0.091; 0.113; 0.155
Report type
Mean
Reported size (nm)
71
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210126 2/9 Homo sapiens Cell culture supernatant (d)(U)C Silva, Andreia;Lázaro-Ibáñez, Elisa 2021 78%

Study summary

Full title
All authors
Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker
Journal
J Extracell Vesicles
Abstract
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide)
EV-METRIC
78% (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
CD47-GFP
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/ CD81/ Alix/ Flotillin1/ CD10
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
80
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
100
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD63/ GFP/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
CD9
Detected contaminants
calnexin
Flow cytometry
Type of Flow cytometry
NanoAnalyzer N30 (nanoFCM)
Calibration bead size
0.068; 0.091; 0.113; 0.155
Antibody details provided?
No
Detected EV-associated proteins
GFP
Detected EV-associated proteins
CD81/ CD9/ CD63/ GFP
Detected EV-associated proteins
GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoAnalyzer N30 (nanoFCM)
Hardware adjustment
Calibration bead size
0.068; 0.091; 0.113; 0.155
Report type
Mean
Reported size (nm)
75
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210126 3/9 Homo sapiens Cell culture supernatant (d)(U)C Silva, Andreia;Lázaro-Ibáñez, Elisa 2021 78%

Study summary

Full title
All authors
Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker
Journal
J Extracell Vesicles
Abstract
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide)
EV-METRIC
78% (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
TSPAN14-GFP
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/ CD81/ Alix/ Flotillin1/ CD11
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
80
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
100
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ Alix/ CD63/ TSG101/ GFP/ CD81
Not detected EV-associated proteins
CD9
Detected contaminants
calnexin
Flow cytometry
Type of Flow cytometry
NanoAnalyzer N30 (nanoFCM)
Calibration bead size
0.068; 0.091; 0.113; 0.155
Antibody details provided?
No
Detected EV-associated proteins
GFP
Detected EV-associated proteins
CD81/ CD9/ CD63/ GFP
Detected EV-associated proteins
GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoAnalyzer N30 (nanoFCM)
Hardware adjustment
Calibration bead size
0.068; 0.091; 0.113; 0.155
Report type
Mean
Reported size (nm)
73
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210126 4/9 Homo sapiens Cell culture supernatant (d)(U)C Silva, Andreia;Lázaro-Ibáñez, Elisa 2021 78%

Study summary

Full title
All authors
Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker
Journal
J Extracell Vesicles
Abstract
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide)
EV-METRIC
78% (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
CD63-GFP
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/ CD81/ Alix/ Flotillin1/ CD12
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
80
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
100
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ Alix/ GFP/ CD63/ TSG101/ CD81
Not detected EV-associated proteins
CD9
Detected contaminants
calnexin
Flow cytometry
Type of Flow cytometry
NanoAnalyzer N30 (nanoFCM)
Calibration bead size
0.068; 0.091; 0.113; 0.155
Antibody details provided?
No
Detected EV-associated proteins
GFP
Detected EV-associated proteins
CD9/ CD63/ GFP
Detected EV-associated proteins
GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoAnalyzer N30 (nanoFCM)
Hardware adjustment
Calibration bead size
0.068; 0.091; 0.113; 0.155
Report type
Mean
Reported size (nm)
74
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210126 5/9 Homo sapiens Cell culture supernatant (d)(U)C Silva, Andreia;Lázaro-Ibáñez, Elisa 2021 78%

Study summary

Full title
All authors
Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker
Journal
J Extracell Vesicles
Abstract
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide)
EV-METRIC
78% (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
CD81TM-GFP
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/ CD81/ Alix/ Flotillin1/ CD13
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
80
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
100
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
CD9
Detected contaminants
calnexin
Flow cytometry
Type of Flow cytometry
NanoAnalyzer N30 (nanoFCM)
Calibration bead size
0.068; 0.091; 0.113; 0.155
Antibody details provided?
No
Detected EV-associated proteins
GFP
Detected EV-associated proteins
CD81/ CD9/ CD63/ GFP
Detected EV-associated proteins
GFP
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoAnalyzer N30 (nanoFCM)
Hardware adjustment
Calibration bead size
0.068; 0.091; 0.113; 0.155
Report type
Mean
Reported size (nm)
74
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210126 6/9 Homo sapiens Cell culture supernatant (d)(U)C Silva, Andreia;Lázaro-Ibáñez, Elisa 2021 78%

Study summary

Full title
All authors
Andreia M. Silva, Elisa Lázaro-Ibáñez, Anders Gunnarsson, Aditya Dhande, George Daaboul, Ben Peacock, Xabier Osteikoetxea, Nikki Salmond, Kristina Pagh Friis, Olga Shatnyeva, Niek Dekker
Journal
J Extracell Vesicles
Abstract
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. Ho (show more...)Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63+, CD81+, or CD9+ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50–170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications. (hide)
EV-METRIC
78% (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
SDCBP-GFP
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/ CD81/ Alix/ Flotillin1/ CD14
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability (%)
80
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
100
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ GFP/ CD63/ TSG101/ Alix/ CD81
Not detected EV-associated proteins
CD9
Detected contaminants
calnexin
Flow cytometry
Type of Flow cytometry
NanoAnalyzer N30 (nanoFCM)
Calibration bead size
0.068; 0.091; 0.113; 0.155
Antibody details provided?
No
Detected EV-associated proteins
GFP