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You searched for: EV220119 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV220119 3/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.07-1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60% (58,7% with sample)
Total gradient volume, incl. sample (mL)
11.97
Sample volume (mL)
0.07
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
169,044
Duration (min)
>960
Fraction volume (mL)
0.997
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
EV220119 4/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.07-1.21
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60% (56.4 % with sample)
Total gradient volume, incl. sample (mL)
4.25
Sample volume (mL)
0.07
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
169,639
Duration (min)
>960
Fraction volume (mL)
0.354
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
EV220119 5/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.07-1.21
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60% (51.8 % with sample)
Total gradient volume, incl. sample (mL)
1.742
Sample volume (mL)
0.07
Orientation
Bottom-up
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
120
Fraction volume (mL)
0.145
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Wide-field
EV220119 6/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.07-1.19
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
1.742
Sample volume (mL)
0.07
Orientation
Top-down
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
120
Fraction volume (mL)
0.145
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV220119 7/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.25-1.11
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
15
Lowest density fraction
0.4M
Highest density fraction
2.5M (2.05M with sample)
Total gradient volume, incl. sample (mL)
1.86
Sample volume (mL)
0.07
Orientation
Bottom-up
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
120
Fraction volume (mL)
0.155
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV220119 8/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.20-1.12
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
15
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
1.86
Sample volume (mL)
0.07
Orientation
Top-down
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
120
Fraction volume (mL)
0.155
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV220119 9/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.17-1.12
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
15
Lowest density fraction
0.4M
Highest density fraction
2.5M (2.05M with sample)
Total gradient volume, incl. sample (mL)
1.86
Sample volume (mL)
0.07
Orientation
Bottom-up
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
810
Fraction volume (mL)
0.155
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV220119 11/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
schistosomula
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.07-1.21
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60% (51.8 % with sample)
Total gradient volume, incl. sample (mL)
1.742
Sample volume (mL)
0.07
Orientation
Bottom-up
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
120
Fraction volume (mL)
0.145
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV220119 12/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
schistosomula
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.07-1.19
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
1.742
Sample volume (mL)
0.07
Orientation
Top-down
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
120
Fraction volume (mL)
0.145
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV220119 13/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
schistosomula
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.26-1.25
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
15
Lowest density fraction
0.4M
Highest density fraction
2.5M (2.05M with sample)
Total gradient volume, incl. sample (mL)
1.86
Sample volume (mL)
0.07
Orientation
Bottom-up
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
810
Fraction volume (mL)
0.155
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV220119 14/14 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers, Marije 2022 56%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
56% (59th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
schistosomula
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
EV density (g/ml)
1.28-1.21
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
15
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
1.86
Sample volume (mL)
0.07
Orientation
Top-down
Rotor type
TLS-55
Speed (g)
166,180
Duration (min)
810
Fraction volume (mL)
0.155
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV220119 1/14 Schistosoma mansoni whole parasite culture (d)(U)C Kuipers, Marije 2022 44%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
44% (12th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Wash: volume per pellet (ml)
11
Wash: time (min)
65
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
96,808
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Extra information
In some cases, 2 collected EV fractions from protocol 3 and 4 were pooled and subjected to a washing step to re-pellet the EVs. Washing UC step of fractions from protocol 3 were performed in SW32 (average 125,755 ×g) and fractions from protocol 4 in SW41 (average 126,444 ×g) for 65 minutes. In another case, the collected fractions from protocol 3, 4 and 5 were subjected to TCA. In addition, in one experiment, adult worm EVs obtained by protocol 5 were concentrated and washed by ultrafiltration (10kDa, regenerated cellulose). Finally, density gradient fractions from protocol 5-10 were directly mixed with sample buffer.
EV220119 10/14 Schistosoma mansoni whole parasite culture (d)(U)C Kuipers, Marije 2022 44%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
44% (12th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
schistosomula
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
96,808
Wash: volume per pellet (ml)
11
Wash: time (min)
65
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
96,808
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV220119 2/14 Schistosoma mansoni whole parasite culture (d)(U)C
UF
qEV
Kuipers, Marije 2022 25%

Study summary

Full title
All authors
Marije E Kuipers, Roman I Koning, Erik Bos, Cornelis H Hokke, Hermelijn H Smits, Esther N M Nolte-'t Hoen
Journal
J immunol Res
Abstract
In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to (show more...)In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources. (hide)
EV-METRIC
25% (3 percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Ultrafiltration
qEV
Protein markers
EV: S. mansoni TSP-2
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Ultra filtration
Cut-off size (kDa)
10 & 3
Membrane type
Regenerated cellulose
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
S. mansoni TSP-2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
1 - 14 of 14
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV220119
species
Schistosoma
mansoni
sample type
whole
parasite
culture
condition
adult
worm
adult
worm
adult
worm
adult
worm
adult
worm
adult
worm
adult
worm
schistosomula
schistosomula
schistosomula
schistosomula
adult
worm
schistosomula
adult
worm
separation protocol
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC/
Density
gradient
dUC
dUC
dUC/
Ultrafiltration/
qEV
Exp. nr.
3
4
5
6
7
8
9
11
12
13
14
1
10
2
EV-METRIC %
56
56
56
56
56
56
56
56
56
56
56
44
44
25