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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
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
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.
    • (d)(U)C = (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
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
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
Density medium
Iodixanol
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
Detected EV-associated proteins
CD9/ ANXA2
Not detected contaminants
APOA1
Detected EV-associated proteins
CD9/ CD63/ ICAM
Not detected contaminants
APOA1
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
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
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
Cell Name
Expi293F
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.
    • (d)(U)C = (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
Sample Condition
CD63-mCherry
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
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
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ mCherry/ Alix/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
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
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
Cell Name
Expi293F
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.
    • (d)(U)C = (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
Sample Condition
CD63-FLuc
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
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
Detected EV-associated proteins
Flotillin1/ Alix/ Firely luciferase/ CD9/ CD63/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
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
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
Cell Name
MDA-MB-468
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.
    • (d)(U)C = (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
Sample Condition
Control condition
EV-producing cells
MDA-MB-468
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
39
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
118000
Density gradient
Density medium
Iodixanol
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
Detected EV-associated proteins
CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5
Detected contaminants
Albumin
Flow cytometry
Hardware adjustments
Proteomics
Proteomics database
Yes: ProteomeXchange
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
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
Cell Name
MDA-MB-468
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.
    • (d)(U)C = (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
Sample Condition
Control condition
EV-producing cells
MDA-MB-468
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
39
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
118000
Density gradient
Density medium
Iodixanol
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
Detected EV-associated proteins
CD9/ CD63/ CD81/ ANXA2/ ANXA5
Not detected EV-associated proteins
Alix/ TSG101
Detected contaminants
Albumin
Flow cytometry
Hardware adjustments
Proteomics
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
EV200185 1/3 Staphylococcus aureus Cell culture supernatant DG
(d)(U)C
Filtration
Bitto, Natalie J. 2021 78%

Study summary

Full title
All authors
Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos
Journal
J Extracell Vesicles
Abstract
Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (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
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
Cell Name
S. aureus NCTC6571
Sample origin
Control condition
Focus vesicles
Other / Membrane vesicles (MVs)
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
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: S. aureus proteins
non-EV: None
Proteomics
no
EV density (g/ml)
1.069-1.119
Show all info
Study aim
Function
Sample
Species
Staphylococcus aureus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
S. aureus NCTC6571
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
P28S
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
20%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.1
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12ml
Pelleting: duration (min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
12
Pelleting-wash: duration (min)
120
Pelleting-wash: speed (g)
SW 40 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
S. aureus proteins
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
Particle yield
particles per colony forming units (CFU) of bacteria;Yes, other: 1010000000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200185 2/3 Staphylococcus aureus Cell culture supernatant DG
(d)(U)C
Filtration
Bitto, Natalie J. 2021 78%

Study summary

Full title
All authors
Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos
Journal
J Extracell Vesicles
Abstract
Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (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
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
Cell Name
S. aureus BPH2760
Sample origin
Control condition
Focus vesicles
Other / Membrane vesicles (MVs)
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
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: S. aureus proteins
non-EV: None
Proteomics
no
EV density (g/ml)
1.069-1.119
Show all info
Study aim
Function
Sample
Species
Staphylococcus aureus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
S. aureus BPH2760
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
P28S
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
20%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.1
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12ml
Pelleting: duration (min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
12
Pelleting-wash: duration (min)
120
Pelleting-wash: speed (g)
SW 40 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
S. aureus proteins
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
Particle yield
particles per colony forming units (CFU) of bacteria;Yes, other: 28000000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200185 3/3 Staphylococcus aureus Cell culture supernatant DG
(d)(U)C
Filtration
Bitto, Natalie J. 2021 78%

Study summary

Full title
All authors
Natalie J Bitto, Lesley Cheng, Ella L Johnston, Rishi Pathirana, Thanh Kha Phan, Ivan K H Poon, Neil M O'Brien-Simpson, Andrew F Hill, Timothy P Stinear, Maria Kaparakis-Liaskos
Journal
J Extracell Vesicles
Abstract
Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to (show more...)Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host. (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
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
Cell Name
S. aureus BPH2900
Sample origin
Control condition
Focus vesicles
Other / Membrane vesicles (MVs)
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
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: S. aureus proteins
non-EV: None
Proteomics
no
EV density (g/ml)
1.069-1.119
Show all info
Study aim
Function
Sample
Species
Staphylococcus aureus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
S. aureus BPH2900
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
P28S
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
20%
Highest density fraction
45%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.1
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12ml
Pelleting: duration (min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
12
Pelleting-wash: duration (min)
120
Pelleting-wash: speed (g)
SW 40 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
S. aureus proteins
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
Particle yield
particles per colony forming units (CFU) of bacteria;Yes, other: 53000000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200118 1/1 Homo sapiens Cell culture supernatant (d)(U)C
ExoQuick
Filtration
UF
Tu, Chenggong 2021 75%

Study summary

Full title
All authors
Chenggong Tu, Zhimin Du, Hui Zhang, Yueyuan Feng, Yujun Qi, Yongjiang Zheng, Jinbao Liu, Jinheng Wang
Journal
Theranostics
Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal cells (BMSCs) have been demonstrated as key factors in the progression and drug resistance of multiple myeloma (MM). EV uptake involves a variety of mechanisms which largely depend on the vesicle origin and recipient cell type. The aim of the present study was to identify the mechanisms involved in the uptake of BMSC-derived small EVs (sEVs) by MM cells, and to evaluate the anti-MM effect of targeting this process. Methods: Human BMSC-derived sEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. The effects of chemical inhibitors and shRNA-mediated knockdown of endocytosis-associated genes on sEV uptake and cell apoptosis were analyzed by flow cytometry. The anti-MM effect of blocking sEV uptake was evaluated in vitro and in a xenograft MM mouse model. Results: sEVs derived from BMSC were taken up by MM cells in a time- and dose-dependent manner, and subsequently promoted MM cell cycling and reduced their chemosensitivity to bortezomib. Chemical endocytosis inhibitors targeting heparin sulphate proteoglycans, actin, tyrosine kinase, dynamin-2, sodium/proton exchangers, or phosphoinositide 3-kinases significantly reduced MM cell internalization of BMSC-derived sEVs. Moreover, shRNA-mediated knockdown of endocytosis-associated proteins, including caveolin-1, flotillin-1, clathrin heavy chain, and dynamin-2 in MM cells suppressed sEV uptake. Furthermore, an endocytosis inhibitor targeting dynamin-2 preferentially suppressed the uptake of sEV by primary MM cells ex vivo and enhanced the anti-MM effects of bortezomib in vitro and in a mouse model. Conclusion: Clathrin- and caveolin-dependent endocytosis and macropinocytosis are the predominant routes of sEV-mediated communication between BMSCs and MM cells, and inhibiting endocytosis attenuates sEV-induced reduction of chemosensitivity to bortezomib, and thus enhances its anti-MM properties. (hide)
EV-METRIC
75% (94th 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
Cell culture supernatant
Cell Name
primary bone marrow stromal cells
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
ExoQuick
Filtration
Ultrafiltration
Protein markers
EV: CD63/ Flotillin1/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary bone marrow stromal cells
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63
Not detected contaminants
Calreticulin
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-210
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 9.00E+07
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-120
EV200013 1/2 Bos taurus Bovine ampulla oviductal fluid DG
qEV
Asaadi, Anise 2021 75%

Study summary

Full title
All authors
Anise Asaadi, Nima Azari Dolatabad, Hadi Atashi, Annelies Raes, Petra Van Damme, Michael Hoelker, An Hendrix, Osvaldo Bogado Pascottini, Ann Van Soom, Mojtaba Kafi, Krishna Chaitanya Pavani
Journal
Int J Biol Sci
Abstract
Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AO (show more...)Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality. (hide)
EV-METRIC
75% (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
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
Bovine ampulla oviductal fluid
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
qEV
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Function
Sample
Species
Bos taurus
Sample Type
Bovine ampulla oviductal fluid
Sample Condition
Control condition
Separation Method
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
14
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
165 +/- 5.8 nm
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.80E+12
EM
EM-type
Transmission-EM
Image type
Close-up
EV200013 2/2 Bos taurus Bovine ovarian follicular fluid DG
qEV
Asaadi, Anise 2021 75%

Study summary

Full title
All authors
Anise Asaadi, Nima Azari Dolatabad, Hadi Atashi, Annelies Raes, Petra Van Damme, Michael Hoelker, An Hendrix, Osvaldo Bogado Pascottini, Ann Van Soom, Mojtaba Kafi, Krishna Chaitanya Pavani
Journal
Int J Biol Sci
Abstract
Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AO (show more...)Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality. (hide)
EV-METRIC
75% (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
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
Bovine ovarian follicular fluid
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
qEV
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Function
Sample
Species
Bos taurus
Sample Type
Bovine ovarian follicular fluid
Sample Condition
Control condition
Separation Method
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
14
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
166.9 +/- 9.7
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 6.44E+12
EM
EM-type
Transmission-EM
Image type
Close-up
EV200010 1/4 Homo sapiens Cell culture supernatant (d)(U)C
SEC
UF
Kuypers, Sören 2021 75%

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
75% (94th 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
Cell culture supernatant
Cell Name
HUVEC
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC
UF
Protein markers
EV: ANXA2/ CD81/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
ANXA2/ CD81/ CD9
Not detected contaminants
GM130
Detected EV-associated proteins
CD9/ CD63/ ICAM1
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
EV200010 2/4 Homo sapiens Cell culture supernatant (d)(U)C
SEC
UF
Kuypers, Sören 2021 75%

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
75% (94th 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
Cell culture supernatant
Cell Name
HUVEC
Sample origin
TNFalpha
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
SEC
UF
Protein markers
EV: ANXA2/ CD81/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
TNFalpha
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
ANXA2/ CD81/ CD9
Not detected contaminants
GM130
Detected EV-associated proteins
CD9/ CD63/ ICAM1
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
EV200010 3/4 Homo sapiens Cell culture supernatant (d)(U)C
SEC
UF
Kuypers, Sören 2021 75%

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
75% (94th 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
Cell culture supernatant
Cell Name
HUVEC
Sample origin
IL1beta
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
SEC
UF
Protein markers
EV: ANXA2/ CD81/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
New methodological development/Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
IL1beta
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
ANXA2/ CD81/ CD9
Not detected contaminants
GM130
Detected EV-associated proteins
CD9/ CD63/ ICAM1
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
EV200007 1/1 Homo sapiens breast milk DG
(d)(U)C
SEC
Zonneveld, Marijke 2021 75%

Study summary

Full title
All authors
Marijke I Zonneveld, Martijn J C van Herwijnen, Marcela M Fernandez-Gutierrez, Alberta Giovanazzi, Anne Marit de Groot, Marije Kleinjan, Toni M M van Capel, Alice J A M Sijts, Leonie S Taams, Johan Garssen, Esther C de Jong, Michiel Kleerebezem, Esther N M Nolte-'t Hoen, Frank A Redegeld, Marca H M Wauben
Journal
J Extracell Vesicles
Abstract
Maternal milk is nature's first functional food. It plays a crucial role in the development of the i (show more...)Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract. (hide)
EV-METRIC
75% (83rd 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
breast 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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
SEC
Protein markers
EV: CD63/ Flotillin1/ CD9/ HSP70
non-EV: Lactoferrin
Proteomics
no
EV density (g/ml)
1.06-1.19
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
breast milk
Sample Condition
Control condition
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
Iodixanol
Type
Continuous
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12.5
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
0.5
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
15
Sample volume/column (mL)
2.5
Resin type
Sephadex G-100
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ HSP70
Detected contaminants
Lactoferrin
Characterization: Particle analysis
EV210044 1/1 Homo sapiens Serum qEV Newman, Lauren 2021 71%

Study summary

Full title
All authors
Lauren A. Newman, Alia Fahmy, Michael J. Sorich, Oliver G. Best, Andrew Rowland, Zivile Useckaite
Journal
Cells
Abstract
Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human bl (show more...)Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a ‘liquid biopsy’ to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease. (hide)
EV-METRIC
71% (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
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
Serum
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
qEV
Protein markers
EV: CD81/ ASGR1/ CD63/ CD9
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Flow cytometry
Type of Flow cytometry
CytoFlex S
Hardware adjustments
1. Filter configuration as per Beckman Coulter website 2. Violet filter used for small particle detection 3. Calibration beads were used to determine particle size gating strategy 4. Callibration beads used Megamix Plus SSC and Megaamix Plus FSC
Calibration bead size
0.1-0.9
Detected EV-associated proteins
CD63/ CD9/ CD81/ ASGR1
Proteomics
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
85.8
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.25E+11
Particle analysis: flow cytometry
Flow cytometer type
CytoFlex S
Hardware adjustment
1. Laser configuration to detect small particles, violet laser use, hardware set up according to Beckman Coulter website 2. Use of MegaMix beads to determine particle size for gating purposes 3. MegaMix plus beads were used: MegaMix Plus SSC and MegaMix Plus Forward FSC
Calibration bead size
0.1-0.9
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200159 1/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 67%

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
67% (92nd 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
Cell culture supernatant
Cell Name
Expi293F
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD63/ Flotillin1/ CD9/ CD81
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
Sample Condition
Control condition
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
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
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ Alix/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
No NA
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
56
EV200157 7/10 Homo sapiens Cell culture supernatant (d)(U)C
SEC (non-commercial)
Polymer-based precipitation
Martínez-Greene, Juan A 2021 67%

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
67% (92nd 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
Cell culture supernatant
Cell Name
MDA-MB-468
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.
    • (d)(U)C = (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
Protein markers
EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5
non-EV: Albumin
Proteomics
no
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
Sample Condition
Control condition
EV-producing cells
MDA-MB-468
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(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
Detected EV-associated proteins
CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5
Detected contaminants
Albumin
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
138
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.91E+11
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200157 8/10 Homo sapiens Cell culture supernatant (d)(U)C
SEC (non-commercial)
Polymer-based precipitation
DG
Martínez-Greene, Juan A 2021 67%

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
67% (92nd 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
Cell culture supernatant
Cell Name
MDA-MB-468
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.
    • (d)(U)C = (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
no
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
Sample Condition
Control condition
EV-producing cells
MDA-MB-468
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(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
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected EV-associated proteins
Alix/ TSG101/ ANXA2/ ANXA5
Detected contaminants
Albumin
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
129
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 5.19E+10
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200157 9/10 Homo sapiens Cell culture supernatant (d)(U)C
SEC (non-commercial)
Polymer-based precipitation
Martínez-Greene, Juan A 2021 67%

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
67% (92nd 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
Cell culture supernatant
Cell Name
gingival primary fibroblasts
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.
    • (d)(U)C = (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
Protein markers
EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5
non-EV: Albumin
Proteomics
no
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
Sample Condition
Control condition
EV-producing cells
gingival primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(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
Detected EV-associated proteins
CD9/ CD63/ CD81/ ANXA2/ ANXA5
Not detected EV-associated proteins
Alix/ TSG101
Detected contaminants
Albumin
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
149
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.89E+11
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200157 10/10 Homo sapiens Cell culture supernatant (d)(U)C
SEC (non-commercial)
Polymer-based precipitation
DG
Martínez-Greene, Juan A 2021 67%

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
67% (92nd 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
Cell culture supernatant
Cell Name
gingival primary fibroblasts
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.
    • (d)(U)C = (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
no
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
Sample Condition
Control condition
EV-producing cells
gingival primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(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
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected EV-associated proteins
Alix/ TSG101/ ANXA2/ ANXA5
Detected contaminants
Albumin
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
158.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.68E+10
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200090 1/2 Homo sapiens Cell culture supernatant (d)(U)C
SEC commercial
UF
Arab, Tanina; Mallick, Emily 2021 67%

Study summary

Full title
All authors
Tanina Arab, Emily R. Mallick, Yiyao Huang, Liang Dong, Zhaohao Liao, Zezhou Zhao, Olesia Gololobova, Barbara Smith, Norman J. Haughey, Kenneth J. Pienta, Barbara S. Slusher, Patrick M. Tarwater, Juan Pablo Tosar, Angela M. Zivkovic, Wyatt N. Vreeland, Michael E. Paulaitis, Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesi (show more...)We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP‐IRIS events could not be used to estimate particle concentrations. For sizing, SP‐IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP‐IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP‐IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single‐particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies. (hide)
EV-METRIC
67% (92nd 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
Cell culture supernatant
Cell Name
promonocytic line U937
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC commercial
UF
Protein markers
EV: CD81/ TSG101/ CD63/ CD9
non-EV: BiP/GRP78/ GM130
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
promonocytic line U937
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
TH-641
Pelleting: speed (g)
110000
Ultra filtration
Cut-off size (kDa)
3
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ CD81
Not detected EV-associated proteins
TSG101/ CD9
Not detected contaminants
BiP/GRP78/ GM130
Fluorescent NTA
Relevant measurements variables specified?
NA
Detected EV-associated proteins
CD81
Not detected EV-associated proteins
CD9/ CD63
Detected EV-associated proteins
CD63/ CD81
Detected EV-associated proteins
CD9/ CD81/ CD63
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
25-250
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.00E+09
EM
EM-type
Transmission-EM
Image type
Wide-field
Report type
Size range/distribution
Report size
25-250
EV-concentration
Yes
Report type
Size range/distribution
Report size
25-250
EV-concentration
Yes
Report type
Size range/distribution
Report size
25-250
EV200090 2/2 Homo sapiens Cell culture supernatant (d)(U)C
SEC commercial
UF
Arab, Tanina; Mallick, Emily 2021 67%

Study summary

Full title
All authors
Tanina Arab, Emily R. Mallick, Yiyao Huang, Liang Dong, Zhaohao Liao, Zezhou Zhao, Olesia Gololobova, Barbara Smith, Norman J. Haughey, Kenneth J. Pienta, Barbara S. Slusher, Patrick M. Tarwater, Juan Pablo Tosar, Angela M. Zivkovic, Wyatt N. Vreeland, Michael E. Paulaitis, Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesi (show more...)We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP‐IRIS events could not be used to estimate particle concentrations. For sizing, SP‐IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP‐IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP‐IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single‐particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies. (hide)
EV-METRIC
67% (92nd 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
Cell culture supernatant
Cell Name
T lymphocyte line H9
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC commercial
UF
Protein markers
EV: CD81/ TSG101/ CD63/ CD9
non-EV: BiP/GRP78/ GM130
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
T lymphocyte line H9
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
TH-641
Pelleting: speed (g)
110000
Ultra filtration
Cut-off size (kDa)
3
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ CD81
Not detected contaminants
BiP/GRP78/ GM130
Fluorescent NTA
Relevant measurements variables specified?
NA
Detected EV-associated proteins
CD81
Not detected EV-associated proteins
CD63
Detected EV-associated proteins
CD81/ CD63
Detected EV-associated proteins
CD9/ CD81/ CD63
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
25-250
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.00E+09
EM
EM-type
Transmission-EM
Image type
Wide-field
Report type
Size range/distribution
Report size
25-250
EV-concentration
Yes
Report type
Size range/distribution
Report size
25-250
EV-concentration
Yes
Report type
Size range/distribution
Report size
25-250
EV200076 1/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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
Cell culture supernatant
Cell Name
Dinoflagellate
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Alix/ HSP70/ beta-actin/ enolase
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/novel EV type
Sample
Species
microalgae
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Dinoflagellate
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
118000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
beta-actin/ enolase/ HSP70/ Alix
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
92
NTA
Report type
Size range/distribution
Reported size (nm)
125
EV concentration
Yes
Particle yield
number of particles per mg dry weight microalgal mass 6.00E+09
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV200076 2/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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
Cell culture supernatant
Cell Name
Diatom
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Alix/ HSP70/ enolase
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/novel EV type
Sample
Species
microalgae
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Diatom
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
118000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
enolase/ HSP70/ Alix
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
135
NTA
Report type
Size range/distribution
Reported size (nm)
90
EV concentration
Yes
Particle yield
number of particles per mg dry weight microalgal mass 2.40E+08
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV200076 3/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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
Cell culture supernatant
Cell Name
Glaucophyte
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Alix/ HSP70/ beta-actin/ enolase
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/novel EV type
Sample
Species
microalgae
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Glaucophyte
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
118000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
118000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ HSP70/ beta-actin/ enolase
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
125
NTA
Report type
Size range/distribution
Reported size (nm)
122
EV concentration
Yes
Particle yield
number of particles per mg dry weight microalgal mass 2.00E+09
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV200076 4/5 microalgae Cell culture supernatant (d)(U)C Picciotto, Sabrina 2021 67%

Study summary

Full title
All authors
Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni
Journal
Biomaterials science
Abstract
Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide)
EV-METRIC
67% (92nd 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