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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
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
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.
    • (d)(U)C = (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
Sample Condition
raw
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
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
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.
    • (d)(U)C = (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
Sample Condition
raw
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
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
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.
    • (d)(U)C = (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
Sample Condition
pasteurized
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
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
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.
    • (d)(U)C = (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
Sample Condition
pasteurized
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
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
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.
    • (d)(U)C = (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
Sample Condition
pasteurized and homogenized
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
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
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.
    • (d)(U)C = (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
Sample Condition
pasteurized and homogenized
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
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
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.
    • (d)(U)C = (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
Sample Condition
UHT
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
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
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.
    • (d)(U)C = (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
Sample Condition
UHT
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Density medium
Sucrose
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFG-E8/ TSG101
Not detected contaminants
beta-casein/ beta-lactoglobulin
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
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